Keely+W.

__**Week 13-15**__

Objective: To make my yopH protein using BL21(DE3) cells for enzyme assays.
 * Protein Expression w/ Surrogate(yopH) - Trial 2**

Induction Step(Option B), then centrifuged the next day using the Beckman centrifuge. Samples were decanted and pellets were weigh to be about 1.2g. Then the pellets were resuspended in 10mL of lysis buffer and transferred to a 15mL conical tube and stored in the -20degC freezer. The next step that will be done will be purification on the yopH protein.


 * Protein Purification w/ Surrogate(yopH)- Trial 2**

Objective: To purify the protein using the affinity tag and Ni-NTA resin.

Fig 1. Absorbance reading of Elution 1 at 280nm for the wavelength using a Nanodrop Spectrophotometer. The concentration of Elution 1 is 1.38mg/ml.

Fig 2. Absorbance reading of Elution 2 at 280nm for the wavelength using a Nanodrop Spectrophotometer. The concentration of Elution 1 is .29mg/ml.

Analysis: The surrogate, yopH was purified by a column chromatography technique with the affinity tag and Ni-NTA resin.The absorbance readings for elution 1 was higher than elution 2 which was predicted due to the extra wash of imidazole. This can either be my protein or contamination. Characterization of the sample collected throughout expression and purification to test that yopH was expressed and purified.


 * Characterization w/ Surrogate(yopH)- Trial2**

Objective: To separate the protein samples by gel electrophoresis and to determine purity and yield.

Fig 3. SDS-PAGE Gel of yopH after expression and purification. To the left is a Bio-rad Molecular Weight Standard for PageRuler Prest. MW standard #26616. Lane 2:Colorplus protein ladder. Lane3: Sample0 containing the cell lysate before induction. Lane4: Sample1-cell lysate after induction. Lane5: Sample2-soluble fraction. Lane6: Sample3- Flow through. Lane7: Sample4- Wash. Lane8: Sample5-Elution1. Lane9: Sample6- Elution2.

Analysis: Figure 3 from characterization showed that yopH was successfully expressed. The molecular weight of yopH is about 33.5Da, which is where the band is located according to the protein ladder. There is a slight smear in lane 8 where the protein is, indicating contamination. Therefore FPLC will be the next step to further purify the protein in order to continue on to enzyme assay.


 * FPLC of yopH:**

Objective: To further purify yopH from the others in the Ni-NTA purified sample, based on the differences in size.

Fig4. Absorbance reading of yopH's elution 1 combined and concentrated with a spin tube concentrator at 280nm for the wavelength using a Nanodrop Spectrophotometer. The concentration of elution1 of yopH is 7.35mg/ml.

Fig5. Absorbance reading of yopH's elution 1 combined after FPLC and non-concentrated at 280nm for the wavelength using a Nanodrop Spectrophotometer. The concentration of elution1 of yopH is .43mg/ml.

Fig6. Absorbance reading of yopH's elution 1 combined after FPLC and concentrated with a spin tube concentrator at 280nm for the wavelength using a Nanodrop Spectrophotometer. The concentration of elution1 of yopH is 1.06mg/ml.

Fig7. Graph of FPLC results with standard curve as the dotted line. The peak from cuvettes 39-44 represents the yopH protein.

Analysis: My elution #1 sample and Caroline's elution #1 sample were combined to get higher concentration and concentrated with a Spin Tube Concentrator as shown in Figure 4. Then combined sample was taken to FPLC for size purification using a buffer(100mM Tris, 150 NaCl, pH 8.0), and the Unicorn program. Figure 7 show the results of FPLC, the peak at cuvette range 39-44 represents the yopH protein. The resulting sample from FPLC was taken to be Nanodropped, which results are shown in Figure5. The concentration was lower because the protein was purified more, getting rid of excess contamination. After FPLC, the samples were concentrated with the concentrator which raised the concentration to 1.06 shown in figure 6. This solution was stored in glycerol in the -20degC freezer, with the next step being an enzyme assay.


 * Enzyme Assay**

Objective: To see if enzyme works.

Fig 8. Results of the Enzyme assay of yopH shown in an Absorbance vs. Enzyme Concentration graph taken at a wavelength of 410nm with error bars that are the Standard Deviations.

Analysis: Once the yopH surrogate was successfully expressed and purified, an enzyme assay was performed to test whether the enzyme worked. The more yopH that was added, the higher the absorbance, making the solution's color turn more yellow after each extra addition of yopH. This indicates that the enzyme worked and about 16-20uL of the enzyme will be diluted and used for the inhibition assay because that amount was where there was the most significant jump in absorbance.


 * Inhibition Assay**

Objective: Inhibition test of compound. Fig9. Results of the first run of the Inhibition assay of yopH shown in an Absorbance vs. Inhibition Concentration graph taken at a wavelength of 410nm and using the inhibitory c ompound 5852635.

Fig10. Results of the second run of the Inhibition Assay of yopH shown in an Absorbance vs. Inhibition Concentration graph taken at a wavelength of 410nm and using the inhibitory compound 5852635.

Analysis: An inhibition assay was performed on the yopH surrogate against the phosphatase inhibitor 5852635. Once the inhibitor was added the absorbance showed a trend of decreasing as shown in both figures 9 and 10(after the third column). The first and second runs showed to be significantly different, indicating that an error was made when making one of the assays. The first run in figure 9 showed the last column to actually increase, which is wrong. This could indicated that we forgot to add the inhibitor. There was a trend of decreasing which is what was suppose to happen, indicating that there was inhibitory effect. However the ratio of enzyme and inhibitory compound could have been off, with too much enzyme added initially. Therefore, when running again, the concentration of the enzyme added would be lowered to produce a more effective inhibitory effect.


 * Virtual Screening**

Objective:T o use GOLD/Hermes to prepare a protein for docking

Fig11. Positive Control Ligand 4 (Endothall) in sticks with carbons colored green. TbPP2B’s active site in sticks with carbons colored cyan blue. The black dashed represent the polar contacts of the Positive Control Ligand 1. Hydrophobic residues are colored red.
 * Table1.** Results of Docking Control ligands in GOLD giving the score, S(PLP), S(hbond), S(cho), S(metal), DE(clash), DE(tors), intcor, polar contacts, and whether they follow Lipinski’s rule. Negative control ligands are highlighted in red, and positive control ligands are highlighted in green.

** Fig12. ** Negative Control Ligand 1 in sticks with carbons colored green. TbPP2B’s active site in sticks with carbons colored cyan blue. The black dashed represent the polar contacts of the Positive Control Ligand 1. Hydrophobic residues are colored red.

**Table2.**
Results of the top 30 compounds in virtual screening using GOLD on Library CB306vsTbPP2B,giving the score, S(PLP), S(hbond), S(cho), S(metal), DE(clash), DE(tors), and intcor.

**Table3**.
Properties of the top 10 compounds virtually screened in the CB306 library in Gold. Properties include: the name, formula, whether it follows Lipinski’s rules, and a 2-D image of the compound.

**Table4.**
Results of the top 30 compounds in virtual screening using GOLD on Library HF9PlatesPlates5_9 vs TbPP2B,giving the score, S(PLP), S(hbond), S(cho), S(metal), DE(clash), DE(tors), and intcor.

**Table5.** Results of the top 4 compounds in virtual screening using GOLD on Library InHouseCompoundsvsTbPP2B,giving the score, S(PLP), S(hbond), S(cho), S(metal), DE(clash), DE(tors), and intcor.
====**Table8.** Top 15 Compounds out of all the virtual screened libraries in Gold, with there rank, score, S(PLP), S(hbond), S(cho), S(metal), DE(clash), DE(tors), Ligand Name, and Library that it was screened are listed for each compound. ====
 * Table6.** Properties of the top 4 compounds virtually screened in the InHouseCompoundsvsTbPP2B library in Gold. Properties include: the name, formula, whether it follows Lipinski’s rules, and a 2-D image of the compound.
 * Table7**. Results of the top 21 compounds in virtual screening using GOLD on Library NIHClinicalCollectionvsTbPP2B,giving the score, S(PLP), S(hbond), S(cho), S(metal), DE(clash), DE(tors), and intcor.

Fig13. Compound SAM001247096 (Salmeterol), #1 Ranked of overall libraries screened,from the NIHClinicalCollection Library in sticks with carbons colored green. TbPP2B’s active site in sticks with carbons colored cyan blue. The black dashed represent the polar contacts of Compound SAM001247096. Hydrophobic residues are colored red.

Fig14. Compound SAM001246840 (Alprostadil), #2 Ranked of overall libraries screened,from the NIHClinicalCollection Library in sticks with carbons colored green. TbPP2B’s active site in sticks with carbons colored cyan blue. The black dashed represent the polar contacts of Compound SAM001246840. Hydrophobic residues are colored red. Fig15. Compound SAM001246678 (Secoisolariciresinol), #3 Ranked of overall libraries screened,from the NIHClinicalCollection Library in sticks with carbons colored green. TbPP2B’s active site in sticks with carbons colored cyan blue. The black dashed represent the polar contacts of Compound SAM001246678. Hydrophobic residues are colored red. Fig16. Compound SAM001247090 (SMR000466290), #4 Ranked of overall libraries screened,from the NIHClinicalCollection Library in sticks with carbons colored green. TbPP2B’s active site in sticks with carbons colored cyan blue. The black dashed represent the polar contacts of Compound SAM001247090. Hydrophobic residues are colored red. Compound SAM001246581 (Valsartan), #5 Ranked of overall libraries screened,from the NIHClinicalCollection Library in sticks with carbons colored green. TbPP2B’s active site in sticks with carbons colored cyan blue. The black dashed represent the polar contacts of Compound SAM001246581. Hydrophobic residues are colored red.
 * [[image:#5.png width="380" height="240"]] Fig17. **

Week 11&12
 * Very good work -UM**
 * Very good work -UM**

Objective: To purify the protein using the affinity tag and Ni-NTA resin.
 * Protein Purification:**

Fig 1. Elution 1 of TbPP2B in a 15mL conical tube Fig 2. Elution 2 of TbPP2B in a 15mL conical tube.

Fig 3. Absorbance reading of Elution 1 at 280nm for the wavelength using a Nanodrop Spectrophotometer. The concentration of Elution 1 is .22mg/ml.

Fig 4. Absorbance reading of Elution 2 at 280nm for the wavelength using a Nanodrop Spectrophotometer. The concentration of Elution 1 is .03mg/ml.

Analysis: My target, TbPP2B was purified by a column chromatography technique with the affinity tag and Ni-NTA resin.The absorbance readings for elution 1 was higher than elution 2 which was predicted due to the extra wash of imidazole. However, the absorbance of elution 1 was relatively small. This can either be my protein or contamination. Characterization is the next step to determining if my protein was expressed and purified.


 * Characterization:**

Objective: To separate the protein samples by gel electrophoresis and to determine purity and yield.

Fig 5. SDS-PAGE Gel of T. brucei protein phosphatase 2B, putative after expression and purification. To the left is a Bio-rad Molecular Weight Standard for PageRuler Prest. MW standard #26616. Lane 2:Colorplus protein ladder. Lane3: Sample0 containing the cell lysate before induction. Lane4: Sample1-cell lysate after induction. Lane5: Sample2-soluble fraction. Lane6: Sample3- Flow through. Lane7: Sample4- Wash. Lane8: Sample5-Elution1. Lane9: Sample6- Elution2.

Analysis: Characterization confirmed that the protein was not pure. Lane 8 should have a distinct band at about 50kDa, which is the size of my target. Instead, there is just a smear representing contamination.There were many steps in the process of expression, purification, and characterization that could have caused errors to have this results. Since the protein failed to purify, expression, purification, and characterization needs to be redone.


 * Virtual Work:**

Objective: To find a set of control ligands and prep them for docking for virtual screening to find potential inhibitors for TbPP2B.
 * Ligand Prep:**

List of Control Ligands used for Virtual Screening for TbPP2B. (CHEMBL122539) pos7 ||  10921447  ||  196.138182  ||  C6H13O5P  ||  -1  ||  3  ||  5  || (CHEMBL123163) pos9 ||  11183084  ||  188.183922  ||  C7H9O2PS  ||  1.6  ||  2  ||  3  || (CHEMBL122938) pos10 ||  13818  ||  96.022362  ||  CH5O3P  ||  -1.6  ||  2  ||  3  || __Analysis:__ 10 positive and 5 negative control ligands were found. Since there are no current inhibitors of TbPP2B reported, I had to do some extensive digging into research articles to find the potential ligands. The first 3 ligands: Okadaic Acid, CsA, and FK506 were specific compounds that I found in literature have shown to be effective for calcineurin. Endothall is a common compound that shows to inhibit various proteins similar to my target. Positive 5-10 ligands were ligands that were inhibitors for serene-threonine phosphates STP1, which has been proven to be similar to my protein. The actual compounds that were found to be inhibitors are very large in size, which could affect the way they will get docked. The others are expected to have some inhibition but not a significant amount. The 5 negative control ligands were found by using the ZINC database. A control ligand library was set up by concatenation with these ligands, and the ligands were prepped in ICM. The next step is prepping the protein and defining the active site.
 * Table 1.**
 * Name ||  ID  ||  Molecular Weight (g/mol)  ||  Molecular Formula  ||  XLogP3-AA:  ||  H-Bond Donor  ||  H-Bond Acceptor  ||
 * Okadaic Acid ||  446512  ||  805.00292  ||  C44H68O13  ||  3.4  ||  5  ||  13  ||
 * CsA(Cyclosporine) ||  5284373  ||  1202.61124  ||  C62H111N11O12  ||  7.5  ||  5  ||  12  ||
 * FK506(Tacrolimus) ||  445643  ||  804.01816  ||  C44H69NO12  ||  2.7  ||  3  ||  12  ||
 * Endothall ||  3225  ||  186.162  ||  C8H10O5  ||  -.5  ||  2  ||  5  ||
 * CHEMBL123495(pos5) ||  10880008  ||  266.186502  ||  C12H11O5P  ||  1.1  ||  3  ||  5  ||
 * CHEMBL122519(SureCN6304059) pos6 ||  10379025  ||  282.252102  ||  C12H11O4PS  ||  2.8  ||  3  ||  5  ||
 * 6-phosphonohexanoic Acid
 * CHEMBL331460 (pos8) ||  11031366  ||  220.248922  ||  C7H9O2PS2  ||  2.4  ||  2  ||  4  ||
 * Benzyl-phosphonothioic acid
 * Methylphosphonic acid
 * AKOS002968708 (Negative1) ||  19166762  ||  225.22118  ||  C9H7NO4S  ||  2.1  ||  2  ||  5  ||
 * AQ-750/42050798 (Negative2) ||  984053  ||  408.49334  ||  C21H16N2O3S2  ||  4.6  ||  0  ||  6  ||
 * AC1MZMY5 (Negative3) ||  3901268  ||  323.81602  ||  C20H18ClNO  ||  5.5  ||  1  ||  2  ||
 * 4,6-dichloro-5-ethylpyrimidin-2-amine (Negative 4) ||  241845  ||  192.04588  ||  C6H7Cl2N3  ||  2.6  ||  1  ||  3  ||
 * Aspirin (Negative5) ||  2244  ||  180.15742  ||  C9H8O4  ||  1.2  ||  1  ||  4  ||

Week 9 &10 __Homology Model:__
 * Very clear and concise. Great analysis. - BN**


 * Objective:** Since there is no crystal structure for //T. brucei// Phosphatase 2B, then a homology model needs to be made. A PDB similar to TbPP2B's is used to create the protein structure that is most similar as possible. This model will be used for future virtual screening in order to asses potential inhibitors of TbPP2B.


 * Swiss-Model Template Identification Tool Results:**
 * [[image:kwTbPP2BSwiss.png width="400" height="263"]]Fig.1 Results of the Swiss-Model top hits for TbPP2B's protein sequence.**


 * [[image:SwissPairwaiseAlignment.png width="400" height="326"]]Fig. 2 Pairwise Alignment of the top hit, 4IL1, to TbPP2B.**


 * Table1.** Molprobity output chart of ICMmodel with added Hydrogens and no added flips
 * All-Atom

Contacts || Clashscore, all atoms: |||| 8.11 || 81st percentile* (N=1784, all resolutions) ||
 * ^  |||||||| Clashscore is the number of serious steric overlaps (> 0.4 Å) per 1000 atoms. ||
 * Protein

Geometry || Poor rotamers || 15 || 4.14% || Goal: <1% || **Table 2.** Molprobity output chart of template 4IL1.pdb with added Hydrogens and no added flips
 * ^  || Ramachandran outliers || 6 || 1.45% || Goal: <0.05% ||
 * ^  || Ramachandran favored || 378 || 91.53% || Goal: >98% ||
 * ^  || MolProbity score ^ |||| 2.41 || 52nd percentile* (N=27675, 0Å - 99Å) ||
 * ^  || C β deviations >0.25Å || 2 || 0.52% || Goal: 0 ||
 * ^  || Bad backbone bonds: || 6 / 1666 || 0.36% || Goal: 0% ||
 * ^  || Bad backbone angles: || 19 / 2079 || 0.91% || Goal: <0.1% ||
 * All-Atom

Contacts || Clashscore, all atoms: |||| 9.15 || 97th percentile* (N=75, 3.00Å ± 0.25Å) ||
 * ^  |||||||| Clashscore is the number of serious steric overlaps (> 0.4 Å) per 1000 atoms. ||
 * Protein

Geometry || Poor rotamers || 29 || 1.54% || Goal: <1% || Protein 4IL1, Crystal Structure of the Rat Calcineurin, was the top hit for TbPP2B. It's pairwise alignment is shown in Fig.2. There were a couple of gaps in the pairwise alignments, having a gap percentage of 2%. There were also quite a few insertions and deletions. It also had a 46% Identity, and was 63% for Positives. This PDB and the protein sequence of TbPP2B was used to make a homology ICMmodel in ICM. The quality of its structure was compared using MolProbity. Its results are shown in Tables 1-2. The clash score for the template was 9.15 and its Molprobity score was 1.99. The clash score for the model was 8.11, and its Molprobity score was 2.41.
 * ^  || Ramachandran outliers || 16 || 0.77% || Goal: <0.05% ||
 * ^  || Ramachandran favored || 1981 || 94.78% || Goal: >98% ||
 * ^  || MolProbity score ^ |||| 1.99 || 99th percentile* (N=3130, 3.00Å ± 0.25Å) ||
 * ^  || C β deviations >0.25Å || 5 || 0.25% || Goal: 0 ||
 * ^  || Bad backbone bonds: || 0 / 8444 || 0.00% || Goal: 0% ||
 * ^  || Bad backbone angles: || 8 / 10534 || 0.08% || Goal: <0.1% ||
 * Analysis:**

My cloning failed, but my partner's, Nicole, was successful. Dr. B told me to go on to protein expression, purification, and characterization using her positive clone. These were her results:
 * Cloning: **


 * [[image:BLASTclone3 toDNAworks.png]]Fig3. ** BLAST of forward read of the DNA sequencing sequence of clone #3 on master plate compared to DNA works sequence of T. brucei protein phosphatase 2B, putative.

Clone 3 of Nicole's cloning was successful and is going to be used for protein expression.
 * [[image:Reverseclone#3BLAST.png]]Fig4. ** BLAST of reverse read of the DNA sequencing sequence of clone #3 on master plate compared to DNA works sequence of T. brucei protein phosphatase 2B, putative.
 * Analysis: **


 * Protein Expression: **

Objective: To make my protein using BL21(DE3) cells for enzyme assays.

 Fig 5. Plate of 10uL of TbPP2B and BL21(DE3) cells after transformation(Day1 of Protein Expression)

Fig 6.Plate of 50uL of TbPP2B and BL21(DE3) cells after transformation(Day1 of Protein Expression)


 * Day 3: Large Culture Step( From Option B):**
 * [[image:Screen Shot 2013-11-03 at 1.26.55 PM.png width="640" height="291"]]Fig. 7** OD600 of bacterial culture with TbPP2B using Vernier Visible Spec(Chipper)

Induction Step(Option A), then centrifuged the next day using the Beckman centrifuge. Samples were decanted and pellets were weigh to be about 3.17g and 3.36g. Then the pellets were resuspended in 10mL of lysis buffer and transferred to a 15mL conical tube and stored in the -20degC freezer. The next step that will be done on Monday is the Lysis step.

Great--organized, clear objectives and analysis, and progression in lab. -U.M. Week 7 & 8


 * __PCR squared:__**

__Objective:__ To make a lot of PCR product for PCR cleanup.

100bp Ladder

PCR^2 Trial 1: **Fig 1.** Gel of PCR squared on a 1% Agarose and 1X TAE gel. Lane 1 was skipped. Lane 2 is 100bp ladder. Lanes 3-6 are PCR squared samples that were used using the secondary PCR samples with 1/2 of the oligo mix and annealing temperature of 61degC. The samples consisted of this secondary samples, 5x rxn buffer, 2mM dNTPs, 20uM Forward primer, 20uM Reverse primer, 122uL of autoclaved water, and 2uL of Q5 Polymerase.

__Analysis:__  PCR squared was done, and as shown in figure 1 did not work. The figure above shows indication of primer dimers. A reason that it didn't work could be because the master mix was not spun down by a vortex like instructed. Another run would be required to determine whether this is the reason that it didn't work.

PCR^2 Trial 2: Fig 2. Gel of PCR squared o<span style="font-family: Arial,Helvetica,sans-serif; font-size: 12px;">n a 1% Agarose and 1X TAE gel. Lane 1 was skipped. Lane 2 is 100bp ladder. Lanes 3-6 are PCR squared samples that were used using the secondary PCR samples with and annealing temperature of 61degC. The samples consisted of this secondary samples, 5x rxn buffer, 2mM dNTPs, 20uM Forward primer, 20uM Reverse primer, 122uL of autoclaved water, and 2uL of Q5 Polymerase.

Analysis: Squared didn't work again and showed primer dimers. Next step is to use my partner's, Nicole's, primers.

PCR^2 Trial 3

Fig 3. Gel of PCR squared o<span style="font-family: Arial,Helvetica,sans-serif; font-size: 12px;">n a 1% Agarose and 1X TAE gel. Lane 1 was skipped. Lane 2 is 100bp ladder. Lanes 3-6 are PCR squared samples that were used using the secondary PCR samples with and annealing temperature of 62degC. The samples consisted of this secondary samples, 5x rxn buffer, 2mM dNTPs, 20uM Forward primer, 20uM Reverse primer, 122uL of autoclaved water, and 2uL of Q5 Polymerase.

Analysis: Showed improvement, but not bright or thick enough band to get a high concentration. I used Nicole's primers, and it worked, indicating my primers were the problem before. Next, I am going to mess with the annealing temperatures.

PCR^2 Trial 4: Fig 4. Gel of PCR squared o<span style="font-family: Arial,Helvetica,sans-serif; font-size: 12px;">n a 1% Agarose and 1X TAE gel. Lane 1 was skipped. Lane 2 is 100bp ladder. Lanes 3-6 are PCR squared samples that were used using the secondary PCR samples with and annealing temperature of 62degC(lanes 3-6) and 63degC(lanes 7-10). The samples consisted of this secondary samples, 5x rxn buffer, 2mM dNTPs, 20uM Forward primer, 20uM Reverse primer, 122uL of autoclaved water, and 2uL of Q5 Polymerase.

Analysis: Lanes 2-6 didn't show up because the gel broke while loading, but the samples in lanes 7, 9-10 showed up a thick bands. Samples with the annealing temperature of 63deg is taken to PCR cleanup.

Fig5. Nanodrop results of PCR cleanup of the PCR squared samples with the annealing temperatures at 63degC. The absorbance reading is 65.5 ng/uL at 260mm path.

Analysis: This was an ok concentration, but I decided to PCR squared again to see if I can get a better concentration.

PCR^2 Trial 5:

Fig6. Gel of PCR squared o<span style="font-family: Arial,Helvetica,sans-serif; font-size: 12px;">n a 1% Agarose and 1X TAE gel. Lane 1 was skipped. Lane 2 is 100bp ladder. Lanes 3-6 are PCR squared samples that were used using the secondary PCR samples with and annealing temperature of 64degC. The samples consisted of this secondary samples, 5x rxn buffer, 2mM dNTPs, 20uM Forward primer, 20uM Reverse primer, 122uL of autoclaved water, and 2uL of Q5 Polymerase.

Analysis: Results didn't come out very well. The bands were faded except for the the few in lanes 8 and 9. I decided to run PCR clean up on the samples in lanes 7-10 and hope to get a decent enough concentration.

Fig7. Nanodrop results of PCR cleanup of the PCR squared samples with the annealing temperatures at 64degC. The absorbance reading is 38.6 ng/uL at 260mm path.

Analysis: The concentration was too low so I decided to go with the PCR cleanup with the 65.5 ng/ul concentration.


 * Transformation of pNIC-Bsa4**

Fig8. pNIC-Bsa4 pellets after centrifugation

__**MidiPrep:**__


 * Midiprep 1:**

Fig8.Nanodrop results of Midiprep. The absorbance reading is 44.5 ng/uL at 260mm path.

Fig9.Nanodrop results of Midiprep. The absorbance reading is 45 ng/uL at 260mm path.

Analysis: Midiprep was successful, giving a decent concentration. When moving on to cutting everything will be increased except for the water to compensate for the concentration.


 * Cutting 1:**

Fig10. Nanodrop results of pNIC-Bsa4 after cutting and cleanup. The absorbance reading is 2.8 ng/uL at 260mm path.

Analysis: Cutting failed, giving me a really low concentration after cleanup of 2.8ng/uL. So transformation of the pNIC-Bsa4 and midi-prep will have to be done again in order to re-do cutting.


 * Midiprep 2**

Fig11.Nanodrop results of Midiprep. The absorbance reading is 45.5 ng/uL at 260mm path.

Fig12. Nanodrop results of Midiprep. The absorbance reading is 45 ng/uL at 260mm path.

Analysis: Midi prep worked giving me a decent concentration. However when moving on to cutting, everything will be increased again except for the water to compensate for the concentration.

Fig13. Nanodrop results of pNIC-Bsa4 after cutting and cleanup. The absorbance reading is 44.3 ng/uL at 260mm path.
 * Cutting2:**

Analysis: Cutting worked with a decent concentration of 44.3ng/ul. Next step cohesion ends then annealing and transformation.

__**Annealing and Transformation:**__ Fig14. Image of the 1:2 ratio plate of pNIC-Bsa4 + TBPP2B for annealing and transformation.

Fig15. Image of the 1:6 ratio plate of pNIC-Bsa4+ TBPP2B for annealing and transformation.

Analysis: Colonies grew!! However I did not a lb+sucrose+kan plate so there is no indication that these were the cloned colonies. Next I will make a master plate on a sucrose plate to see if anything shows up.

Master Plate Fig16. Image of the Master Plate for pNIC-Bsa4 + TBPP2B

Analysis: Nothing showed up on my master plate. This could be because the colonies from annealing and transformation were not plasmid with my target because sucrose wasn't used when sucrose was used in this plate. This means I have to start all the way back to PCR squared then midi prep, cutting, and annealing transformation to redo the master plate.

I felt like I was reading a blog... You must've been here a lot. I'm glad you finally got PCR working. Keep up the good work! Awesome job! - Michael T. Week 5 & 6

Objective: To dilute the primers that were ordered for cloning.
 * Primer Dilution for Secondary PCR:**

__TBPP2B- R:__ 31.9nMoles -> .0319 uMoles -> 319uL TE __TBPP2B-F:__ 34.4nMoles -> .0344uMoles -> 344uL TE __Working Dilution:__ 100uM x unknown = 20uM x 200uL unknown= 40uL

__** Secondary PCRs: **__

__Objective:__ To successfully amplify our gene of interest from primary PCR using tail primers to be later used for PCR squared that will synthesize our gene of interest.

__**Trial 1:**__ <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">**Fig 1.** Gel of Secondary PCR on a 1% Agarose and 1X TAE gel. Lane 1 was skipped. Lane 2 and 6 are 1kb Ladders. The 1kb Ladder is shown to the left. Secondary PCR samples include: 31.5 uL of autoclaved water, 5uL of dNTP, 1uL primary PCR, 1uL of each Forward and Reverse Primer that has been diluted, 10uL 5X Reaction Buffer, and .5 uL of Hotstart Q5 Polymerase. Lane 3 is 1 <span style="font-family: Arial,Helvetica,sans-serif;">st <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;"> sample of secondary PCR using the primary PCR in the previous gel. Lane 7 is the 2 <span style="font-family: Arial,Helvetica,sans-serif;">nd <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;"> sample of secondary PCR using the primary PCR from the previous gel.

<span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">Analysis: <span style="font-family: Arial,Helvetica,sans-serif; font-size: 12px;">Secondary from the figure above was run using Primary PCR from the previous gel. Secondary PCR did not work. A reason for this is that when the samples were put into the PCR machine, the machine was not closed all the way. Therefore, it didn’t amplify the DNA. Another error was that the dNTP was not diluted and 10mM of dNTP was used instead of 2mM dNTP. Also, the 1kb ladder was used instead of the preferred 100bp ladder, so it is hard to tell if it actually worked. Sample 1 and 2 had bands that appeared, this could indicate some of the primary PCR that was in the sample that had already been amplified. Since the PCR machine didn’t actually run on the samples, this run on Secondary PCR with the same conditions need to be done again.

__**Trial 2:**__ <span style="font-family: Arial,Helvetica,sans-serif; font-size: 12px; line-height: 0px; overflow: hidden;"> <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">**Fig2.** Gel of the 2nd attempt of Secondary PCR on a 1% Agarose and 1X TAE gel. Lane 1 was skipped. Lane 2 and 6 are the 100bp Ladders. The scale for the 100bp Ladder is on the left. In lane 3 and 7 are the samples of Secondary PCR. Secondary PCR samples include: 31.5 uL of autoclaved water, 5uL of 2mM dNTP, 1uL primary PCR, 1uL of each Forward and Reverse Primer that has been diluted, 10uL 5X Reaction Buffer, and .5 uL of Hotstart Q5 Polymerase. Lane 3 is 1st sample of secondary PCR using the primary PCR in the previous gel. Lane 7 is the 2nd sample of secondary PCR using the primary PCR from the previous gel.

__Analysis:__ <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">The 2nd trial of Secondary PCR did not work. Since there was no indication that the conditions didn’t work on the previous PCR because the 1st trial didn’t actually run, the same conditions were applied. The Secondary sample in lane 3 in the figure above had a strong band with a smear, and lane 7 of the 2nd sample had a slight smear. This looked similar to primary PCR, indicating that secondary PCR didn’t work. A possibility for this is that the Forward and Reverse tail primers didn’t latch on. When preparing the sample, the primers could have not been pipetted properly into the sample. From here, temperatures and times need to be adjusted in the next Secondary PCR run as well as a new primary needs to be made.

__**Trial 3:**__
 * Conditions Changed for Trial 3: **
 * Remade Primary PCR
 * Annealing time change to 30 seconds.

<span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">**Fig 3.** Gel of the 3rd Trial of Secondary PCR on a 1%Agarose and 1XTAE gel. Lane 1 is skipped. Lane 2 and 6 are 100bp Ladders. The scale for 100bp Ladder is on the left. In lane 3 and 7 is primary PCR. Lane 4 and 8 is Secondary PCR. Secondary PCR samples include: 31.5 uL of autoclaved water, 5uL of 2mM dNTP, 1uL primary PCR, 1uL of each Forward and Reverse Primer that has been diluted, 10uL 5X Reaction Buffer, and .5 uL of Hotstart Q5 Polymerase. The Primary PCR used in secondary PCR samples is new and shown in lanes 3 and 7.

__<span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">Analysis: __

<span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">The 3rd trial for Secondary PCR did not work. A new primary PCR was run on the same gel as the secondary PCR. From the image above, the Primary PCRs for lanes 3 and 7 did not appear, indicating that primary PCR did not work, so therefore secondary would not work. However, my partner’s, Nicole, had a slight band that showed her sample on the right track. So next the annealing time will still be increased to 30 second, but the temperature for that time will be changed to a higher and lower temperature.

__**Trial 4:**__ <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">**Fig4.** Gel of 4 <span style="font-family: Arial,Helvetica,sans-serif;">th <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;"> trial of Secondary PCR of Phosphatase 2B from T. brucei on a 1%agarose and 1XTAE gel. Lane 2 and 6 are 100bp Ladders. The scale for 100bp Ladder is on the left. In lane 3 and 7 is primary PCR. Lane 4 and 8 is Secondary PCR with different annealing temperatures. Lane 4’s was run at 55degC, and lane 8’s was run at 62degC. Secondary PCR samples include: 31.5 uL of autoclaved water, 5uL of 2mM dNTP, 1uL primary PCR, 1uL of each Forward and Reverse Primer that has been diluted, 10uL 5X Reaction Buffer, and .5 uL of Hotstart Q5 Polymerase. The Primary PCR used in secondary PCR samples is new and shown in lanes 3 and 7.
 * Conditions Changed from NEB guidelines for Trial 4: **
 * Remade Primary PCR
 * Annealing time changed to 30 seconds
 * Annealing temperature adjusted to a 55 degrees Celsius for one sample and 62 degrees Celsius for the other sample.

__<span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">Analysis: __

<span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">Attempt 4 did not work. Primary was remade for secondary, the annealing time was adjusted to 30 seconds, and the annealing temperature was tweaked at a higher and lower temperature. Lane 4 was the secondary sample with the annealing temperature changed to 55degC, and lane 8 was the secondary sample with the annealing temperature changed to 62degC. Primary PCR was intensified, and the secondary samples appeared to have primer dimers. This indicates that the primers did not attach. Technical malfunctions occurred throughout the time that the gel was run, so another gel using the same samples need to be run again.

__**<span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">Trial 5: **__

<span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">**Fig5.** Gel of the 5th trial of Secondary PCR and Primary PCR of Phosphatase 2B from T. brucei on an agarose gel. Lane 2 is a 100bp Ladder. The scale of 100bp ladder is on the left. Lanes 3-4 are Primary PCR, lanes 5-6 were my partner’s secondary PCR, and lanes 7-8 are my secondary PCR. The same samples were run from the previous figure. Lanes 6 and 8 are the secondary samples with a higher annealing temperature of 62degC, and lanes 5 and 7 were secondary with a lower annealing temperature or 55degC. Secondary PCR samples include: 31.5 uL of autoclaved water, 5uL of 2mM dNTP, 1uL primary PCR, 1uL of each Forward and Reverse Primer that has been diluted, 10uL 5X Reaction Buffer, and .5 uL of Hotstart Q5 Polymerase.

__Analysis:__ Another gel was run on the same samples as the ones in Trial 4, because there was a machine malfunction in the middle of running. This time my partner and I ran it on the same gel. My secondary PCR showed similar results as in the previous gel. It showed indication of primer dimer. Nicole’s PCR samples were a little more encouraging. Although her secondary sample in lane 5 with a lower annealing temperature of 55degC didn’t show up, the sample in lane 6 showed a slight band. This indicates that samples at the higher annealing temperature worked better. But, it is not intense enough. This could be because of the results from primary PCR. As shown in lanes 3-4, the primary samples were really intense and didn’t have the smear that primary should. Next, primary needs to be redone and prove that it is working by running a gel on it. Then using it for secondary where a temperature gradient will be tried starting temperature around 62degC with mostly temperatures above it since the melting temp for our target is in the high 60s-low 70s.


 * Primary PCR with NEB guidelines: **

** Fig6. ** Gel of Primary PCR of Phosphatase 2B from T. brucei on an agarose gel. Lane 2 is a 100bp Ladder, and the scale for 100bp ladder is on the left for reference. Lanes 3-4 are partner’s primary samples, and lanes 5-6 are my primary samples. Primary includes: 10uL 5X buffer reaction, 5uL of 2mM dNTP, 33.5uL autoclaved water, 1uL oligo mix, and .5uL Hotstart (Q5 Polymerase).

__ Analysis: __ From the results of the figure above, primary is no longer working. The bands were too concentrated near the area of the size of our DNA. This was made the same with the same conditions as the 1st trial of primary PCR that worked before. The only possible cause that we could think of was that our oligo mix has denatured since then. We decided to run a secondary PCR with these samples to see if there is any difference. We also plan to make a new oligo mix, and see if that makes a difference.

__** Trial 6: **__


 * Conditions Changed from NEB guidelines for Trial 6: **

Temperature gradient for annealing step: 65degC, 64.5degC, 63degC, and 61.5degC

** Fig7. ** Gel of trial 6 of Secondary PCR from Phosphatase 2B of T. brucei on an agarose gel. Lane 2 is a 1kb Ladder (because there was no more 100bp Ladders). A scale for this ladder is on the left for reference. Lanes 2-6 are samples of secondary PCR using the Primary PCR from the previous gel. Lane three contains secondary PCR at the annealing temperature of 65 degC. Lane four contains secondary PCR at the annealing temperature of 64.5. Lane five contains secondary PCR run at the annealing temperature of 63 degC. Lane six contains secondary PCR run at the annealing temperature of 61.5 degC. All secondary PCR’s other times and temperatures were kept at the NEB guidelines. Secondary PCR samples include: 31.5 uL of autoclaved water, 5uL of 2mM dNTP, 1uL primary PCR, 1uL of each Forward and Reverse Primer that has been diluted, 10uL 5X Reaction Buffer, and .5 uL of Hotstart Q5 Polymerase.

__Analysis:__

Secondary did not work yet again. This trial was done using the primary PCR whose gel was shown in the previous figure. Since the primary of that gel didn’t work well, this secondary PCR working was skeptical. The sample in lane 5 showed the most promise by having a slight band, but it was not as concentrated we want it to be. This trial was not successful and showed even more that the primary was too concentrated and didn’t work. A new oligo mix is the next step to get Primary to work, then to eventually get secondary PCR to work!

__**3rd Primary:**__


 * Conditions Changed from the Protocol for Primary PCR with new Oligo Mix: **
 * A sample made with half the amount of oligo mix and taq (.5uL oligo and .25uL taq)
 * A sample with normal amounts.

**Fig8.** Gel of Primary PCR with the new oligo mix from Phosphatase 2B of T. brucei on an agarose gel. Lane 2 is a 100bp Ladder, and a scale of this ladder is on the left for reference. Lanes 3-4 are primary samples using the regular amounts of oligo and Hotstart(1uL oligo and .5uL Hotstart). Lanes 5-6 are secondary samples using half the amount of oligo mix and Hotstart from the protocol (.5uL oligo and .25 Hotstart). Primary includes: autoclaved water, dNTP, 5X reaction buffer, oligo mix, and Q5 Hotstart. Lane 3 and 5 are my partner’s samples, and lanes 4-6 are my samples.

__ Analysis: __ Primary PCR with the new oligo mix did not turn out as well as expected. Since the previous primary PCR results came back too concentrated, so samples with half the amount of oligo mix and taq was made as well as samples with the normal amounts. The results came back a little too concentrated, but it is better than the previous primary. The most promising primary was my partner’s on lane 5 with half of the oligo and taq. The smear looked good. My samples came back too concentrated again. Which doesn’t make sense since my samples were made in the exact same conditions with the same oligo mix and taq. Next secondary will be run with these samples with a temperature gradient for the annealing time.

__**Trial 7:**__

**Fig9**. Gel of Trial 7 of Secondary PCR of Phosphatase 2B of T. brucei on an agarose gel. Lane 2 is the new 100bp Ladder with its scale on the left. Lane 3 is secondary PCR with the primary sample with half the oligo and taq with the annealing temperature at 62degC. Lane 4 is the secondary sample with the primary sample that had the normal amounts and an annealing temperature at 62degC. Lane 5 is the secondary sample with the primary sample with half the oligo and taq with the annealing temperature at 63degC. Lane 6 is the secondary sample with the primary sample with normal amounts and an annealing temperature at 63degC. Lane 7 is the secondary sample with the primary sample with primary sample with half the oligo and taq and an annealing temperature at 64degC. Lane 8 is the secondary sample with the primary sample with normal amounts and an annealing temperature at 64degC. Lane 9 is the secondary sample with the primary sample with half of the oligo and taq and an annealing temperature at 61degC. Lane 10 is the secondary sample with the primary sample with normal amounts and an annealing temperature at 61degC.

__Analysis:__ Secondary finally worked!!! After the many trials of secondary, we got it to work! In this trial we used the primary from the new oligo mix that I made, and adjusted the amount of oligo and taq when making the primary samples. A temperature gradient was done as well for the secondary PCR. The temperature started at 61degC – 64degC because from previous runs, it worked the best around 62degC. Multiple samples were made, hoping that one of them would worked. From the results of the figure above, all the samples worked but just had a little contamination. The bands were strong, especially at 61 degrees Celsius. The best sample was in lane 5 with the primary with half the oligo and taq and annealing temp of 63 degrees. This sample didn’t have as strong of a band as the others but didn’t have the contamination that the others did as well. The reason that the bands are located so far up compared to the ladder was because this was a new 100bp Ladder that only goes to 1000bp. Knowing this, the results match up to our target because the size of our target is larger than 1000bp. From these results, it can be concluded that our oligo mix was the problem with the other secondary PCRs. Next, PCR squared will be done as well as prepping the pNIC.

Week 3 & 4 Keely - great work. Include a ladder image. Dr. B 092713 ps - HF - high fidelity (vs high frequency)


 * redo of PCR Trial 2 &3 :**

Objective: To successfully amplify the purple protein coding sequence in the pGBR22 plasmid using both the reverse and forward primers.

__Results:__ Fig 1. Gel of PCR samples A-D on a 1% agarose in 1X TAE with a 100bp ladder(Lane 1). Samples A-C represent the different concentrations of plasmid pGBR22. Sample A in lane 2 is a 1:1000 dilution with a 3ng total amount. Sample B in lane 3 is a 1:100 dilution with 3ng total amount. C in lane 4 is a 1:10 dilution with a 30ng total amount. Lane 5 is the Control with no DNA.
 * Trial 2**

Fig 2. Gel of PCR samples A-D on a 1% agarose in 1X TAE with a 100bp ladder(Lane 1). Samples A-C represent the different concentrations of plasmid pGBR22. Sample A in lane 2 is a 1:1000 dilution with a 3ng total amount. Sample B in lane 3 is a 1:100 dilution with 3ng total amount. C in lane 4 is a 1:10 dilution with a 30ng total amount. Lane 5 is the Control with no DNA.
 * Trial 3**

Analysis of Trial 2: From the gel shown in figure 1, the purple protein coding sequence was not successfully amplified into the pGBR22 plasmid. There were no bands other than the 100bp ladder that showed up. Errors that could have occurred were denaturation or contamination of the samples. From here, another redo for this PCR would be required.

Analysis of Trial 3: From the gel shown in figure 2, the purple protein coding sequence was successfully amplified into the pGBR22 plasmid. The band was thicker for sample B which technically shouldn't have happened because it was the second most diluted. Whereas sample C in lane 4 was less diluted and should have been thicker. Nothing should have shown up in lane 5 with sample D because there was no DNA in the sample. However indication of DNA was produced in a very faded band. This could indicate contamination during handling the samples.

__**RE Digest:**__

Objective: In this lab the goal it to digest pGBR22 plasmid with restriction enzymes and visualize fragments on gel. Restriction enzymes EcoRI and PvuII from the Analyzing DNA sequence lab were used.

Results: .Figure1. Gel of RE digest with 1%agarose and 1X TAE. Lane 1 was skipped and the 1kb ladder was used in Lane 2. Lane 3 is the uncut plasmid, 4 is EcoRI, 5 is PvuII, and 6 is the EcoRI+PvuII.

Fig 2. Virtual gel readings for pGBR22 plasmid. From the left is the 1 kb ladder, next is reading of pGBR22 with EcoRI digests, then pGBR22 plasmid with PvuII digests, then pGBR22 plasmid with both EcoRI and PvuII.

Analysis: Since the High Frequency restriction enzymes, EcoRI and PvuII, are being used, NEBuffer 4 was the appropriate buffer. It is recommended to incubate in temperatures between 65 degrees Celsius to 80 degrees Celsius. The same buffer will work for both enzymes. The conditions to stop the EcoRI-HF enzyme are at 65 degrees Celsius for 20 minutes and 80 degrees Celsius for 20 minutes for the PvuII restriction enzyme. According to the RE digest gel shown in figure 1, the restriction enzymes were successfully digested into the pGBR22 plasmid.The bands were compared to the virtual gels from the Analysis of the DNA sequence lab. The uncut plasmid is further down on the ladder than the EcoRI because it is smaller and more coiled, causing it to run faster down the gel. The lane with EcoRI+PvuII (lane 6) should have 3 bands appear. Two bands appeared clearly while the 3rd band is slightly cured. The other samples (Eco+PvuII) appeared clear and aligned with the virtual gel representations(Figure2). Possible contamination and denaturation could have been errors in this lab. From this lab, the DNA sequence can be verified and then the gene of interest can be inserted into the vector.

__**Primary Overlap:**__

__Objective__: The purpose of this lab was to synthesize the oligo mix of all the primers and Nanopure water to store for future procedures for the target gene, //T. brucei's// Phosphatase 2B, putative

__Analysis:__ The oligo mix for Phosphatase 2B required 32 primers(32 wells). The primer plate was left out to thaw beforehand. 68 micro liters of autoclaved water were added to a tube with 1 micro liters of each well to get a total volume of 100 micro liters. This was then stored in the -20 degrees Celsius freezer for later use.

__** PCR Primer Design Tails for pNIC-Bsa4 Cloning :**__

__Objective:__ In this lab, a forward and reverse primer will be designed for my gene of interest sequence in order to amplify the CDS of this gene using PCR. Compatible Ends will be made for proper Ligation Cloning (LIC) to insert into the accepting vector, pNIC-Bsa4, for eventual expression.

__CDS Sequence of //T. brucei's// Phosphatase 2B, Putative:__ TACTTCCAATCCATGTCTTCTGGCGCGTCCCATCATGAGCTGACCCGTGGTCGTGATGGTCTCAAAGAACGTGAATACGTTTGGAAAAAATCTCACGCGGACGAGTCCCCGGGTTCTAACCCGCAAATCCTGCCGACCCTGGTTCTGCCGCACCACCTCGTGTTCGACAACGACGGTGCGCCACTGGCGGACAACATCAAAGTTCACTTCGGTCGCGGTTGGCGCCTCCACGTTGAGGATGCGCTGAATATCGTTCACCGTTGCGCGCTGATCATGAAGGAGGAACCGAATGTTGTACGCCTGAAGGGCTCTGCAGTAGTCTGCGGTGATCTCCACGGCCAGTTCCACGACCTCCTGACCCTGCTGGAAGTTAATGGTCACCCTAGCGTTCAACAGTACGTTTTCCTGGGTGACTACGTTGACCGTGGTGACTTCTCTGCTGAAATCGTTCTGCTGTGCATGTCTTTCAAACTGCTGTACCCGCGCTCTTTCATCCTGCTGCGTGGTAACCACGAGTCTCGCCAGCTGACGTCTTGCTTCAACTTCAAACAGGAAATCGAATCTAAATACTCTTCTATGGTTTACGAAGAAATCATGGCGGCGTTCGACTGCTTCCCGCTGTCTTGCGTTGTTAACGACCGTTTCTTTTGTGTTCATGGTGGTCTCTCTCCGCTGCTGACCTACCTCGGTGAGATCGATACTGTTAACCGTTTTCGTGAAACCCCGTCTACTGGTCCGATGTGTGACCTGCTCTGGTCTGATCCAATGTTCGGTGATGACACCGACTGTGCGACGCCGAGCGAAGAACTGTTCGTTTTTAACACTAAACGTGGTTGCTCTTACAACTACAGCTACGAAGCCGTTTGCCGTTTCCTCGAAGCGAACAATCTGTGCACGGTTATTCGTGGTCATGAGACCCAGCCGGGTGGTTATAAACTGTACCGCCATACCCCAAAGGGTGTTCCGGCGGTTGTATGTGTTTTTAGCGCGAGCAATTATTGCGGTACCTACGGTAACATGGCCGCAGTTGTAGCGATCGATGGTGACGTTATGAACATCCGTCAGTACATGGCGACCTCTCACGACTCTTGCACCCCTAACCACTTCAATGCGATCTCTCGTATGCAGCCTCTGGCGATCCATGAGGCGGTAGAAAAGTGGTGTGTTGCGTCTCACGGTGGTTCTTCCGGTGACGCGAAAGCGGAAAAAGTTGAGGTTGAAAAAAACCTGTCTGAAGATGACAGCTCTGTTGTTCTGCGTGAAAAACTGGGCGATATGATCTGCGCAATGCACCACATTGTTACCTAACAGTAAAGGTGGATA

__Forward Primer:__ TACTTCCAATCCATGTCTTCTGGCGCGTCC (30bp)

GC Content: 53.3%

0 mM Mg2+ Tm: 65.2 oC

1.5 mM Mg2+ Tm: 71.7 oC

2 mM Mg2+ Tm: 72.2 oC

4 mM Mg2+ Tm: 73.1 oC

6 mM Mg2+ Tm: 73.6 oC

__ Reverse Primer: __

CACCACATTGTTACCTAACAGTAAAGGTGGATA

__ Reverse Complement: __

TATCCACCTTTACTGTTAGGTAACAATGTGGTG (33bp)

GC Content: 42.4%

0 mM Mg2+ Tm: 62.7 oC

1.5 mM Mg2+ Tm: 70.2 oC

2 mM Mg2+ Tm: 70.7 oC

4 mM Mg2+ Tm: 71.7 oC

6 mM Mg2+ Tm: 72.2 oC

__**<span style="font-family: Arial,Helvetica,sans-serif;">Sequence of pNIC28 with Phosphatase 2B inserted into the Vector: **__ <span style="font-family: Arial,Helvetica,sans-serif; font-size: 80%;">TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGCACCATCATCATCATCATTCTTCTGGTGTAGATCTGGGTACCGAGAACCTGTACTTCCAATCCATGTCTTCTGGCGCGTCCCATCATGAGCTGACCCGTGGTCGTGATGGTCTCAAAGAACGTGAATACGTTTGGAAAAAATCTCACGCGGACGAGTCCCCGGGTTCTAACCCGCAAATCCTGCCGACCCTGGTTCTGCCGCACCACCTCGTGTTCGACAACGACGGTGCGCCACTGGCGGACAACATCAAAGTTCACTTCGGTCGCGGTTGGCGCCTCCACGTTGAGGATGCGCTGAATATCGTTCACCGTTGCGCGCTGATCATGAAGGAGGAACCGAATGTTGTACGCCTGAAGGGCTCTGCAGTAGTCTGCGGTGATCTCCACGGCCAGTTCCACGACCTCCTGACCCTGCTGGAAGTTAATGGTCACCCTAGCGTTCAACAGTACGTTTTCCTGGGTGACTACGTTGACCGTGGTGACTTCTCTGCTGAAATCGTTCTGCTGTGCATGTCTTTCAAACTGCTGTACCCGCGCTCTTTCATCCTGCTGCGTGGTAACCACGAGTCTCGCCAGCTGACGTCTTGCTTCAACTTCAAACAGGAAATCGAATCTAAATACTCTTCTATGGTTTACGAAGAAATCATGGCGGCGTTCGACTGCTTCCCGCTGTCTTGCGTTGTTAACGACCGTTTCTTTTGTGTTCATGGTGGTCTCTCTCCGCTGCTGACCTACCTCGGTGAGATCGATACTGTTAACCGTTTTCGTGAAACCCCGTCTACTGGTCCGATGTGTGACCTGCTCTGGTCTGATCCAATGTTCGGTGATGACACCGACTGTGCGACGCCGAGCGAAGAACTGTTCGTTTTTAACACTAAACGTGGTTGCTCTTACAACTACAGCTACGAAGCCGTTTGCCGTTTCCTCGAAGCGAACAATCTGTGCACGGTTATTCGTGGTCATGAGACCCAGCCGGGTGGTTATAAACTGTACCGCCATACCCCAAAGGGTGTTCCGGCGGTTGTATGTGTTTTTAGCGCGAGCAATTATTGCGGTACCTACGGTAACATGGCCGCAGTTGTAGCGATCGATGGTGACGTTATGAACATCCGTCAGTACATGGCGACCTCTCACGACTCTTGCACCCCTAACCACTTCAATGCGATCTCTCGTATGCAGCCTCTGGCGATCCATGAGGCGGTAGAAAAGTGGTGTGTTGCGTCTCACGGTGGTTCTTCCGGTGACGCGAAAGCGGAAAAAGTTGAGGTTGAAAAAAACCTGTCTGAAGATGACAGCTCTGTTGTTCTGCGTGAAAAACTGGGCGATATGATCTGCGCAATGCACCACATTGTTACCTAACAGTAAAGGTGGATACGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATTGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTTCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGATCATGCGCACCCGTGGGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGTCGAGATCTCGATCCCGCGAAAT

Fig 1. Virtual Gel of pNic28-Bsa4 with the enzyme BsaI (Left), next is the virtual gel of CDS of Phosphatase 2B from //T. brucei// with enzyme BsaI, then on the right is the virtual gel of the CDS of Phosphatase 2B from //T. brucei// inserted in the CDS pNIC28-Bsa4 with the enzyme BsaI.

__Analysis:__ In this lab, the CDS of our gene of interest, Phosphatase 2B, was used to find the tail primers to be used in further procedures.The CDS of Phosphatase 2B was inserted into the vector pNIC28-Bsa4 and the enzyme BsaI was analyzed for cutting. The G.C content as well as the specific melting temperatures in which different concentrations of Mg were found for both the forward and reverse primer. This information will be used in future labs when working with my target. Both the forward and reverse complement tail primers were ordered for future labs.

__**Primary PCR:**__

Objective : The goal of this lab was to make the Primary PCR solution using the diluted oligo mix that was previously prepared using the primers that were ordered for //T. brucei's// Phosphatase 2B, putative. A gel was then run to verify the solution.

Fig 1. Gel of Primary PCR with 1%agarose in 1XTAE with a 1kb ladder. Two samples were run. The first sample is is lane 3 with the 1kb ladder in lane 2. The second sample of the primary PCR is in lane 8 with the 1 kb ladder in lane 7.

__Analysis:__ Primary PCR was done using the solution of: 5X rxn buffer, diluted dNTP, our oligo mix, dH2O and Q5 hotstart polymerase.Two samples were made using the bother the higher and lower ends of the cycle. The PCR was run overnight using the NEB guidelines, then later stored in the -20 degrees Celsius freezer. A gel was then run to check the solution using a 1 kb ladder.The first sample didn't show up, but the second sample appeared as a smeared band. This smear was predicted and verified that the solution was correct. a reason why the first sample didn't appear could be that it wasn't inserted properly into the well, because the loading was not inserted with the sample until the sample was added to the well. Sample 2 however was inserted correctly and the results appeared correct. The next step after this is secondary PCR.

Week 1 & 2 Keely, good work. Redo PCR (and you can use the smaller well combs for your gel) Dr. B 090913


 * Quantifying DNA using Nanodrop**

Objective: The purpose of this experiment is to quantify the DNA sample, pGBR22, by using Nanodrop spectrophotometry.

Fig.1 Nanodrop results for measurement 1 of pGBR22 with a concentration of 202.8 ng/uL.

Fig 2. Nanodrop results for measurement 2 of pGBR22 with a concentration of 206.8 ng/uL.

Analysis/Conclusion: pGBR22 was quantified by Nanodrop spectrophotometry. The average concentration for both measurements was 204.8 ng/uL, which was close to the original concentration of 205.4 ng/uL. The sample was verified as pure by their 260/280 and 260/230 values, signifying that there was no contaminants. Errors that could have occurred during this experiment could be not properly blanking the instrument, or wiping off the pedestal before taking a measurement. Another error that could have skewed the results were using a contaminated sample to measure. From here, pGBR22 can be prepared for submitting the DNA to the DNA sequencing facility for a primer to be added.


 * Submitting DNA to DNA Sequencing Facility:**

Objective: The purpose of this lab was to prepare the DNA template pGBR22 to be submitted to the core facility for DNA sequencing with a primer added.

Fig. 1 Form for DNA sequencing to core facility for primer M13F to be added and sequencing to be done on pGBR22.

__The DNA sequence results:__ NNNNNNNNNNNNNNGGCGATTGGGNNNACGTCGCATGCTCCCGGCCGCCATGGCCGCGGGATTTTAGTGATGGTGATGGTGATGACCGAGCAAAGAGTGGCGTGCAATGGATATTTCACACTGCTCAACAAATGTGTAATCCTTGTTGTGACTGGTTACATCCAGTTTGCGGTCAACATAGTGATACCCTGGCATCCTCACAGGCTTCTTTGCCTTGTAAGTAGATTTGAATTCACACAAATAGTAACCACCTCCTTCCAACTTCAGAGCCATAAAGTTGTTTCCTATCAGCATTCCATCTCGTGCAAAGAGACGCTCAGTGTTGGGTTCCCAGCCCTGTGTCTTCTTCTGCATAACAGGTCCATTGGGAGGAAAGTTCACACCAGAGATTTTGACATTGTAGATGAAACAGTTGCCTTGGATGCTGGAATCATTGCTGACAGTACACACTGCACCATCTTCAAAGTTCATGATCCTCTCCCATGTATATCCCTCAGGGAATGACTGCTTTACATAATCAGGGATGTCTTCAGGGTACTTGGTGAATGGTATGCTTCCGTATTGAGACAGTGGTGATAAAATATCCCAAGCAAATGGCAGAGGTCCACCCTTGGTGACAGTGAGCTTTACCGTCTGCTCCCCCTCGTAAGGCTTTCCTTTTCCATCGCCTTCGACCTCAAAGTAGTGTCCATTGACCGTGCCTGACATATAAACCTTGTANGTCATTTGTTTAGCGATCACACTCATGATATTTCTCCTTCAATCAATCAAAATCACTAGTGCGGCCGCCTGCNNNCGACCATATGGGANAGCTCCCAACGCGTTGGATGCATAGCTTGAGTATTCTATAGTGTCACCTAAATAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCNCACAACATACGAGCCGGAAGCNTAAAGTGTAAAGCCTGGGGNGCCTAATGAGTGANCTAACTCNCATTAATTGCNTTGCNNNNNCTGCCCGCTTTCCAGTCNGGAANNCTGTCGTGCCNGCTGCATNATGATNNNNNNCNNNGGNNNNNNNNGNTNGCGTANNNGGNNNTNNNNNNNTCCTCNNTNNNNGANTCNNTGNNNNNNGTCNTNGNNGCGGNNNNNNGNNNNNNNNNNNNNTNNNNGNNNNNCGNNNNCCNNNNANNNGNNNNNNNNCNNNAANANNNNNNNNNNNNAANNNNNNNNNNNNNNNNCNNNNNNNCNNNNNNNNGNNNTTTNNNNNNNNNNNNNCCCNNNNNNNNNNNNNNNNN

Fig 2. Nucleotide BLAST of the DNA sequence of pGBR22 with M13F against Human with all nucleotide collection databases.

__Analysis/Conclusion:__ The DNA template pGBR22 was prepared and submitted to the ICMB Core Facilities for DNA sequencing. The in-house primer M13F was chosen to be added by the core. After settting up an account and preparing the template, the template was submitted the next day to the core facility. Results were posted hours later. The errors that could have occurred in this lab could be not having the right temperature for the primer or DNA template, or not adding the right amount of DNA template when preparing for submitting. The next step after this is analyzing the DNA sequence results.

__**PCR Primer Design for Primer Overlap Assembly PCR:**__

Objective: The purpose of this experiment was to design a set of oligo primers for PCR synthesizing for cloning. code Job started on 09/02/2013 at 15:56:33

Job name: KW090213Tbruceiphosphatase2B

Output will be sent to keely_shaylyn31@hotmail.com

SEQUENCE 1: PROTEIN LENGTH =  431 1 MSSGASHHELTRGRDGLKEREYVWKKSHADESPGSNPQILPTLVLPHHLVFDNDGAPLAD 61 NIKVHFGRGWRLHVEDALNIVHRCALIMKEEPNVVRLKGSAVVCGDLHGQFHDLLTLLEV 121 NGHPSVQQYVFLGDYVDRGDFSAEIVLLCMSFKLLYPRSFILLRGNHESRQLTSCFNFKQ 181 EIESKYSSMVYEEIMAAFDCFPLSCVVNDRFFCVHGGLSPLLTYLGEIDTVNRFRETPST 241 GPMCDLLWSDPMFGDDTDCATPSEELFVFNTKRGCSYNYSYEAVCRFLEANNLCTVIRGH 301 ETQPGGYKLYRHTPKGVPAVVCVFSASNYCGTYGNMAAVVAIDGDVMNIRQYMATSHDSC 361 TPNHFNAISRMQPLAIHEAVEKWCVASHGGSSGDAKAEKVEVEKNLSEDDSSVVLREKLG 421 DMICAMHHIVT

Sequence Patterns Screened (As Supplied By User) code code

None found

32 oligonucleotides need to be synthesized 1 ATGTCTTCTGGTGCCTCTCATCACGAGCTGACGCGTGGCCGTGACGGTCTGAAAG 55 2 GAGATTCGTCCGCGTGGGATTTTTTCCAAACGTATTCACGCTCTTTCAGACCGTCACGGC 60 3 CCACGCGGACGAATCTCCGGGTTCTAACCCGCAGATCCTGCCGACCCTGGTTCTGCCGCA 60 4 TGTTATCGGCGAGCGGGGCACCATCGTTGTCGAACACCAGGTGATGCGGCAGAACCAGGG 60 5 CCCGCTCGCCGATAACATCAAAGTTCACTTTGGTCGCGGCTGGCGTCTGCACGTCGAGGA 60 6 TTCCTTCATGATGAGCGCGCAACGGTGAACGATGTTGAGCGCATCCTCGACGTGCAGACG 60 7 CGCGCTCATCATGAAGGAAGAACCGAACGTTGTTCGTCTCAAAGGTTCCGCTGTTGTTTG 60 8 CAGGGTGAGCAGGTCGTGGAACTGACCATGCAGGTCACCGCAAACAACAGCGGAACCTTT 60 9 ACGACCTGCTCACCCTGCTGGAAGTCAACGGTCACCCGTCTGTTCAACAGTACGTGTTTC 60 10 TCCGCAGAGAAGTCACCACGGTCCACATAATCACCGAGAAACACGTACTGTTGAACAGAC 60 11 TGGTGACTTCTCTGCGGAAATCGTTCTGCTCTGCATGAGCTTCAAACTGCTGTACCCGCG 60 12 AGTTGACGAGACTCGTGGTTACCACGCAGCAGGATGAAGCTACGCGGGTACAGCAGTTTG 60 13 ACCACGAGTCTCGTCAACTGACGTCTTGCTTCAATTTCAAGCAGGAGATCGAATCTAAAT 60 14 AACGCCGCCATGATTTCTTCGTAAACCATAGAAGAGTATTTAGATTCGATCTCCTGCTTG 60 15 AAGAAATCATGGCGGCGTTCGACTGCTTCCCGCTCTCCTGTGTTGTTAACGACCGTTTCT 60 16 GGTAGGTCAGCAGCGGAGAGAGACCACCGTGAACGCAGAAGAAACGGTCGTTAACAACAC 60 17 TCCGCTGCTGACCTACCTGGGTGAAATCGACACTGTTAACCGTTTTCGTGAAACGCCGTC 60 18 ACATCGGGTCAGACCACAGCAGATCACACATCGGACCGGTAGACGGCGTTTCACGAAAAC 60 19 TGTGGTCTGACCCGATGTTCGGCGACGACACCGATTGCGCGACCCCGTCCGAAGAACTGT 60 20 AAGAGTAGTTGTAGCTGCAACCACGTTTAGTGTTAAAAACGAACAGTTCTTCGGACGGGG 60 21 GGTTGCAGCTACAACTACTCTTACGAGGCTGTATGCCGCTTCCTGGAAGCGAACAATCTG 60 22 ACCACCCGGTTGCGTCTCGTGACCACGGATAACGGTACACAGATTGTTCGCTTCCAGGAA 60 23 GACGCAACCGGGTGGTTATAAACTGTACCGCCACACCCCGAAGGGTGTTCCGGCGGTAGT 60 24 GTTACCGTAGGTACCGCAGTAATTAGATGCGGAGAAAACGCACACTACCGCCGGAACACC 60 25 CTGCGGTACCTACGGTAACATGGCAGCGGTTGTTGCGATCGACGGTGACGTTATGAATAT 60 26 GGGTGCAAGAATCGTGGGAGGTCGCCATGTACTGACGGATATTCATAACGTCACCGTCGA 60 27 CCCACGATTCTTGCACCCCTAATCACTTCAACGCGATCTCTCGTATGCAGCCACTGGCCA 60 28 CCGCCGTGAGATGCAACACACCACTTTTCTACCGCTTCGTGGATGGCCAGTGGCTGCATA 60 29 GTTGCATCTCACGGCGGCAGCTCTGGTGACGCGAAGGCTGAAAAGGTCGAAGTCGAAAAA 60 30 CACGCAGGACTACAGAGGAGTCATCTTCAGACAGGTTTTTTTCGACTTCGACCTTTTCAG 60 31 TCCTCTGTAGTCCTGCGTGAGAAACTGGGCGACATGATCTGTGCAATGCATCACATTGTT 60 32 GGTAACAATGTGATGCATTGCACA 24

FINAL SUMMARY FOR  1 SOLUTION

#   Tm   Len  |    Score   TmRange  Short    Long   #Olig  #Repeat #Misprime

1   62    60  |    0.000       1.9      16      60      32       0       0

code Fig 1. Results of the LogFile of T. brucei phosphatase 2B, putative

Analysis/Conclusion: The amino acid sequence for the target T. brucei phosphatase 2B, putative from the TDR website was used to determine the oligoneleotides, or primers, that need to be ordered. There was an error that caused a second run to be made. The wrong YP number was given on the wikispaces page. This caused a wrong amino acid sequence to be run. From here, the next step is ordering the primers and waiting for them in the mail.

__**Analyzing DNA Sequence:**__

Objective: The purpose of this lab was to determine the DNA sequence of a plasmid using a BLAST comparisons.

Fig 1. Screenshot of part of the results of Analyzing DNA sequence, the complete results are found on Google Docs.

__**PCR:**__

Objective: The purpose of this lab is to amplify the purple protein coding sequence in the pGBR22 plasmid using both the reverse and forward primers.

Fig 1. Gel of pGBR22 plasmid samples A-D from PCR. The 1st well is the 100bp marker ladder to be used to evaluate the samples. Wells 2-5 are samples A-D with sample A starting in well 2 and continuing chronologically to the left. Samples A-C are represent the different concentrations of plasmids while sample D does not have any DNA in it.


 * Analysis/Conclusion:**

The pGBR22 plasmid was amplified using PCR with different concentrations in samples A-C and no DNA in sample D. To check to see if the PCR was done successfully, a gel was run. The 100bp marker ladder was used to analyze the results. However after the gel was run, the results were inconclusive because no bands showed up except for the ladder. This signifies that PCR was not done successfully. Possible errors that could account for this could be leaving the plasmid out too long where it denatured or contamination during preparation of the samples. The next step after this would be to re-do the PCR and gel until the results come out successfully.