Aditi+S.

Figure 1: Top 20 ligands from Virtual Screening in GOLD for the target protein
 * Week 15**
 * # Score S(PLP) S(hbond) S(cho) S(metal) DE(clash) DE(tors) intcor time File name Ligand name ||
 * 73.07 || -69.16 || 2 || 0 || 0 || 0 || 1.27 || 0.46 || 15.728 || 'output_103_204/gold_soln_cb_306_3d_m127_2.sdf' || '6956655' ||
 * 66.66 || -65.75 || 0.43 || 0 || 0 || 0 || 0.57 || 0.67 || 31.062 || 'output_103_204/gold_soln_cb_306_3d_m145_2.sdf' || '7558664' ||
 * 66.15 || -62.01 || 1.43 || 0 || 0 || 0 || 0.13 || 0.1 || 13.072 || 'output_205_306/gold_soln_cb_306_3d_m209_3.sdf' || '7766330' ||
 * 64.82 || -65.2 || 1 || 0 || 0 || 0 || 2.19 || 1 || 28.698 || 'output_1_102/gold_soln_cb_306_3d_m83_8.sdf' || '5627579' ||
 * 64.74 || -59.09 || 1.99 || 0 || 0 || 0 || 0.19 || 0.04 || 16.246 || 'output_103_204/gold_soln_cb_306_3d_m203_2.sdf' || '7748043' ||
 * 64.65 || -59.65 || 1.98 || 0 || 0 || 0 || 0.49 || 0.03 || 27.589 || 'output_103_204/gold_soln_cb_306_3d_m201_4.sdf' || '7746693' ||
 * 64.57 || -63.23 || 1 || 0 || 0 || 0 || 0.85 || 0.04 || 19.294 || 'output_103_204/gold_soln_cb_306_3d_m137_9.sdf' || '7493042' ||
 * 64.52 || -62.1 || 0.99 || 0.32 || 0 || 0 || 0.8 || 0.05 || 21.003 || 'output_103_204/gold_soln_cb_306_3d_m184_4.sdf' || '7722615' ||
 * 64.44 || -63.16 || 1.28 || 0 || 0 || 0 || 1.32 || 0.1 || 23.602 || 'output_103_204/gold_soln_cb_306_3d_m135_5.sdf' || '7474459' ||
 * 64.42 || -68.41 || 0 || 0 || 0 || 0 || 2.08 || 0.17 || 39.292 || 'output_1_102/gold_soln_cb_306_3d_m85_9.sdf' || '5654317' ||
 * 64.02 || -61.77 || 1 || 0 || 0 || 0 || 1.45 || 2.15 || 18.634 || 'output_205_306/gold_soln_cb_306_3d_m206_8.sdf' || '7757183' ||
 * 63.75 || -57.28 || 2.39 || 0 || 0 || 0 || 0.35 || 0.01 || 6.431 || 'output_1_102/gold_soln_cb_306_3d_m20_2.sdf' || '5158497' ||
 * 63.43 || -61.4 || 1 || 0 || 0 || 0 || 0.6 || 0.04 || 8.151 || 'output_103_204/gold_soln_cb_306_3d_m158_2.sdf' || '7637949' ||
 * 63.35 || -64.42 || 0 || 0 || 0 || 0 || 0.62 || 0.04 || 9.427 || 'output_205_306/gold_soln_cb_306_3d_m276_1.sdf' || '9027311' ||
 * 63.09 || -60.8 || 0.89 || 0 || 0 || 0 || 0.22 || 0.03 || 6.5 || 'output_205_306/gold_soln_cb_306_3d_m227_1.sdf' || '7870889' ||
 * 62.81 || -63.73 || 1 || 0 || 0 || 0 || 1.98 || 0.04 || 21.686 || 'output_103_204/gold_soln_cb_306_3d_m143_1.sdf' || '7543758' ||
 * 62.5 || -58.88 || 1.76 || 0 || 0 || 0 || 0.85 || 0.06 || 29.767 || 'output_205_306/gold_soln_cb_306_3d_m223_10.sdf' || '7848095' ||
 * 62.33 || -60.59 || 0.99 || 0 || 0 || 0 || 0.68 || 0.02 || 5.607 || 'output_205_306/gold_soln_cb_306_3d_m221_1.sdf' || '7829885' ||
 * 62.31 || -52.25 || 4.02 || 0 || 0 || 0 || 1.13 || 0.12 || 27.75 || 'output_103_204/gold_soln_cb_306_3d_m187_1.sdf' || '7726180' ||
 * 62.11 || -60.32 || 1 || 0 || 0 || 0 || 0.64 || 0.07 || 6.909 || 'output_103_204/gold_soln_cb_306_3d_m157_1.sdf' || '7635462' ||
 * 62.11 || -60.32 || 1 || 0 || 0 || 0 || 0.64 || 0.07 || 6.909 || 'output_103_204/gold_soln_cb_306_3d_m157_1.sdf' || '7635462' ||



Figure 2: Enzyme Assay results. The largest absorbance reading was at an enzyme concentration of 0.63.



Figure 1: Sample containing abs value of 1.647 at 410nm.

Analysis: yopH surragote protein was used to run an enzyme assay and inhibition assay after it was properly expressed and purified. As more yopH was added to the solution more activity was oberserved because the solution turned more and more yellow after the addition of NaOH and there was an increase in absorbance measured for each solution with more yopH concentration. This being said the enzyme assay worked and the 10uL amount of yopH enzyme dilution will be used for inhibition assay because there is a significant jump from 5 uL to 10 uL and should properly be inhibited at this protein amount. As seen in Figure 2, t he largest absorbance reading was at an enzyme concentration of 0.63. A graph of the top twenty ligands from virtual screening is also given in Figure 3.


 * Week 13 & 14 **

Protein Expression:

The positive clone was used to grow up in BL21(DE3) cells that were brought up to the correct concentration, harvested, lysed, spun down, and centrifuged. Samples 1 and 2 were collected throughout this process to be used for protein characterization, but first the final sample collected is purified.

Protein Purification: Figure One: Nanodrop of T. cruzi serine threonine protein phosphatase catalytic subunit, putative protein from the colony after expression and purification (1st elution wash). Concentration of protein after first elution is 0.12 mg/uL.

Figure Two: Nanodrop of T. cruzi serine threonine protein phosphatase catalytic subunit, putative f rom the colony after expression and purification (2nd elution wash). Concentration of protein after second elution is 0.06 ng/uL.

Analysis: Protein sample from expression was purified with the help of a Ni-NTA resin attaching to the 6X his tag on the protein and only releasing when imidazole is present. So theoretically if it worked all of the protein should be in sample 5 ( first elution wash). According to the nanodrop the concentration of sample five is around 1.0 ng/uL. which can either be my protein or contamination. There was some concentration in sample six so not all of the protein was released after 1st elution or is once again contamination. Then I proceeded to characterization.

Protein Characterization

Figure One: SDS-PAGE Gel of T. cruzi serine threonine protein phosphatase catalytic subunit, putative after expression and purification. Lane two: Colorplus protein ladder. Lane three: sample one containing protein after large culture overnight (lysate after induction). Lane four: sample two containing protein pre-syringe filtration (soluble fraction). Lane five: sample three containing flow through. Lane six: sample four containing wash. Lane seven: sample five containing first elution. Lane eight: sample six containing second elution.

Analysis: After expression and purification the protein is pure. Lane five shows a distinct band at roughly the size of my protein. In lane three there is a distinct band where the protein is.

Figure One: BLAST of DNA sequencing sequence of clone on master plate compared to DNA works sequence of T. cruzi serine threonine protein phosphatase catalytic subunit, putative. This is the forward read of the DNA sequencing sequence.
 * Pictures of virtual work? Could use more results. -UM**
 * Week 11 & 12**

Figure Two: BLAST of DNA sequencing sequence of clone on master plate compared to DNA works sequence of T. cruzi serine threonine protein phosphatase catalytic subunit, putative. This is the reverse read of the DNA sequencing sequence.

Virtual Progress: A homology model was made for T. cruzi serine threonine protein phosphatase catalytic subunit, putative using the PDB website. P ositive control ligands and negative controls ligands have also been found using the binding database, Pubchem, and ZINC.

Figure Three: Homology Model of T. cruzi serine threonine protein phosphatase catalytic subunit, putative.



Table 1: List of the ten positive control ligands found on PubChem and the three negative control ligands found on ZINC.


 * Week 9 & 10**
 * Great amount of results for these weeks. Missing sample ID name for the nanodrop results. - BN**


 * MidiPrep **

Objective: The purpose of this lab is to extract pNIC-Bsa4 from previously transformed bacterial cells.

Figure One: Nanodrop of pNIC-Bsa4 in DH5alpha from transformed bacterial cells after midiprep. Concentration is 64.9 ng/uL

Analysis: In this experiment transformed pNIC-Bsa4 in DH5alpha was used to midiprep - a process that extracts the DNA of pNIC-Bsa4 from the transformed bacterial cells.The protocol stated the A-260 10mm path value should be around one, and the value collected, as shown in Figure 1, exceeded one which is good because pNIC-Bsa4 was not only successfully transformed but it was also successfully extracted from the transformed bacteria.


 * Cloning T. cruzi serine threonine protein phosphatase catalytic subunit, putative in pNIC-Bsa4**

Objective: The objective of cutting pNIC-Bsa4 is to prepare the DNA as an accepting vector to insert the target into it.
 * Cutting**

Figure One: Nanodrop of new pNIC-Bsa4 in DH5alpha after PCR cleanup in preparation to be an accepting vector. Concentration is 61.3 ng/uL.

Analysis: In this experiment the solution made from midiprep was used. The pNIC-Bsa4 was cut using NEB Buffer 4, 100x BSA, and BsaI-HF in order to prepare pNIC-Bsa4 as an accepting vector that can accept the target for cloning. About 7 ng/uL of the sample (pNIC-Bsa4) from midiprep was lost during PCR clean up. This was fine since the concentration of pNIC-Bsa4 goes down a little after cutting and clean up, because the clean up is removing any contamination. Nothing was changed procedure wise during transformation, midiprep, or cutting. A new PCR clean up kit was used.


 * Cohesive End Generation, Transformation and Annealing Trial One:**

Objective: The purpose of this lab is to prepare insert and accepting vector for successful cloning. Figure One: T. cruzi serine threonine protein phosphatase catalytic subunit, putative with pNIC-Bsa4 on an LB+KAN (no suc) plates after overnight in the incubator.

Analysis: Although there were colonies on the plates both 1:2 and 1:3 ratios of vector to insert, LB+Kan+Suc plates were not used so pNIC-Bsa4 also grew with our DNA because sucrose kills pNIC. But there were some big colonies so putting a variety of colonies on a master plate with sucrose should show if our DNA grew.


 * Master Plate:**

Objective: The purpose of making a master plate is to join insert and accepting vector to later get positive matches from DNA sequencing.

Figure One: T. cruzi serine threonine protein phosphatase catalytic subunit, putative with accepting vector on a master plate with LB+Kan+Suc, grown overnight in the incubator.

Analysis: On the plate rows 1 and 3 is the 1:2 ratio alternating between big colony and little colony and on rows 2 and 4 the ratio 1:3 plates was used also alternating between big colony and little colony. It is good that the larger colonies grew on the plates because the smaller colonies from the first round of plates were supposed to be pNIC-Bsa4 because sucrose plates weren't used and the larger colonies were supposed to be our DNA. Because colonies grew the next step is to send the transformed tubes to DNA sequencing to check for matches.

Figure One: Nanodrop of T. cruzi serine threonine protein phosphatase catalytic subunit, putative from colony #1 on master plate. Concentration of colony #1 is 161.2 ng/uL.

Figure Two: Nanodrop of T. cruzi serine threonine protein phosphatase catalytic subunit, putative from colony #3 on master plate. Concentration of colony #3 is 154.6 ng/uL.

Figure Three: Nanodrop of T. cruzi serine threonine protein phosphatase catalytic subunit, putative from colony #5 on master plate. Concentration of colony #5 is 157.2 ng/uL.

Figure Four: Nanodrop of T. cruzi serine threonine protein phosphatase catalytic subunit, from colony #7 on master plate. Concentration of colony #7 is 159.3 ng/uL.

Then prepared these solutions and send to DNA sequencing. Waiting for Results


 * Gel Extraction:**

Objective: The purpose of gel extraction is to reduce contamination of DNA by extracting from gel.

Figure One: Gel Check of s erine/threonine protein phosphatase catalytic subunit, putative of PCR squared (from secondary PCR at 63 degC for annealing temperature) to be used for gel extraction.

Figure One: Nanodrop of half of DNA extracted from agarose gel. Concentration of DNA is 70.9 ng/uL.
 * After gel extraction:**

Analysis: After gel extraction (process where bands were cut from gel and purified using kit) the concentration of our DNA was determined using nanodrop to be around 71 ng/uL which is good value for the concentration to be proceeding into cloning. This will be used as the insert for cloning.


 * Good work. Could have more data. -UM**
 * Week 7 & 8**


 * PCR Squared**

Objective: The purpose of PCR squared is to make an excess of PCR since much of it will be lost during PCR clean up.



Figure 1: P CR squared of s erine/threonine protein phosphatase catalytic subunit, putative on an agarose gel. Lane one contains a 1 kb ladder. Lanes three, five, seven, and nine contain the PCR squared solution. PCR squared solution contains diluted dNTP, secondary PCR from last trial, forward and reverse tail primers, reaction buffer, Q5 hotstart and autoclaved water. Analysis: After the secondary PCR worked, PCR squared was also successful. As seen in Figure 1, four distinct bands appeared at the right spot on the gel. There was also some contamination, but this was merely due to an excess of forward or reverse primers in the solution. Now the nex step of PCR clean up could be completed.


 * PCR Clean-Up**

Objective: The purpose of PCR clean up was to purify the PCR squared sample by removing everything from it except for the DNA. This was done to be able to measure an accurate concentration of the DNA.

Figure Two: Nanodrop of s erine/threonine protein phosphatase catalytic subunit, putative after PCR clean-up. Determined concentration to be 49.8 ng/uL.

Analysis: PCR cleanup gave good results because the nanodrop found the DNA's concentration to be 49.8 ng/uL which is close to the actual concentration of s erine/threonine protein phosphatase catalytic subunit, putative. During the process of PCR clean up wash solution and elution buffer were added and centrifuged several times so that the DNA was able to separate completely from all the other solutions in the PCR squared solution. By the end only the DNA was left at the bottom of the tube and the concentration was then determined to be 49.8 ng/uL. This concentration is a little low but this might be because not all of the other substances were all filtered out which could have resulted in the DNA to be more diluted than it should be. The experiment worked overall and now the research can proceed on to the next step of cloning.


 * LB agar plate or LB media **

Objective: The purpose of this protocol was to make 160mL of LB media and four agar plates using LB broth for future experiments, such as growing pNIC-Bsa4 and cloning.

Analysis: LB broth was made and autoclaved and then four LB agar plates were made using 80mL of autoclaved LB broth. The plates and the LB broth were stored in the the fridge. Antibiotic was not added into the LB media or the agar plates.


 * Transformation of competent cells for plasmid prep of pNIC-Bsa4 **

Objective: The purpose of this lab is to transform bacteria using E.coli DH5α and pNIC-Bsa4 to make more plasmid DNA.

Figure 1: Frozen pNIC-Bsa4 pellets

Analysis: On the first day, the bacteria was incubated on LB agar plates for approximately 25 hours. The next day, one bacteria colony was collected by a pipette tip and placed into LB media which was finally placed in 37 degree shaker at 250 rpm for about 16 hours. On the last day, four tubes containing the bacteria and LB media were spun down in the 4 degree centrifuge at 6000 x g for 15 mins, and then the waste liquid was eluted. The bacteria pellet was kept in -20 degree freezer.


 * Good job with the analysis and captions. I'm digging the progress. On to the next one! - Michael T.**
 * Week 5 & 6**

Objective: The purpose of secondary PCR is to take 1 uL of primary PCR that worked properly, run it through the PCR machine again with the forward and reverse tail primers and then run it on an agarose gel, where a thick band (with no contamination) should appear that is the size of the target protein. This secondary PCR will later be used for PCR squared. Figure 1: Secondary PCR and Primary PCR of s erine/threonine protein phosphatase catalytic subunit, putative on agarose gel. Lane 1 is a 1kb ladder, lane 3 is Primary PCR, and lane 5 is Secondary PCR.
 * Secondary PCR**

Figure 2: 1kb ladder used in the gel above Analysis: After Primary PCR was successful, Secondary PCR was made using 1 uL of the primary PCR that worked properly, 5X rxn buffer, diluted dNTP, the newly made oligo mix, diluted forward and reverse primers, dH2O, and Q5 hotstart polymerase. This solution and run through the PCR machine with the NEB recommend guidelines, and then through the agarose gel. Similar to Figure 2 under Primary PCR, Figure 1 does not look as clear as it should, due to the camera, not the actual gel. When looked at through the window the gel was very clear, had no contamination, and the ladder was much more distinct, unlike the picture that was captured. Since the Secondary PCR was successful, the next step is PCR squared.

Figure 3: 1kb ladder used in gels below.
 * Primary PCR** (Two tries)

Figure 2: Primary PCR of s erine/threonine protein phosphatase catalytic subunit, putative on agarose gel. Lane 1 is a 1kb ladder and Lane 3 is Primary PCR.

Figure 1: Primary PCR of s erine/threonine protein phosphatase catalytic subunit, putative on agarose gel. Lane 1 is a 1kb ladder and Lane 3 is Primary PCR.

Analysis: The first time Primary PCR was done, nothing showed up on the gel. The second time the experiment was done with better technique and the same Primary PCR mix, yet nothing showed up again. The third time, the oligo mix was redone and the Primary PCR was remade using 5X rxn buffer, diluted dNTP, our oligo mix, dH2O and Q5 hotstart polymerase, and ran through the PCR machine again using the NEB recommended guidelines. This time a smear appeared on the gel, and preparations for Secondary PCR were then begun. Though Figure 2 does not look as clear as it should, this is due to the camera, not the actual gel. When looked at through the window the gel was very clear, had no contamination, and the ladder was more distinct, unlike the picture that was captured.


 * Week 3 & 4**
 * Aditi - ok good, include analysis after your caption (not within the caption). -- DR. B 092513**


 * Primary PCR**

Objective: The purpose of this lab was to make a primary PCR solution with our target's diluted oligoprimers and see if it showed up on an agarose gel.

Figure 1: Primary PCR of s erine/threonine protein phosphatase catalytic subunit, putative on agarose gel. The gel did not work and no DNA is visible in any of the Lanes. Figure 2: 1kb Ladder used in gel above.

Analysis: In this experiment a primary PCR solution was made using 5X rxn buffer, diluted dNTP, our oligo mix, dH2O and Q5 hotstart polymerase and run through the PCR machine with the NEB recommend guidelines. The gel of the primary PCR did not work because there was nothing showing in any of the lanes. This may have been due to the fact that different primers require different temperatures.


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

__Purpose__: Design a forward and a reverse primer for PCR amplifying the CDS of your gene of interest sequence and synthesizing Compatible Ends suitable for Ligation Independent Cloning (LIC) for insertion into the pNIC-Bsa4 as the accepting vector for eventual expression.

__ Coding DNA Sequence: __

ATGTCCCTCGACGTTGACGCGATCATCGATAAACTGCTGGGTGTTCGTGTTTCTAAACCGGGTAAACAAGTTAATCTGGCAGAAAATGACGTTAAAAACCTGGCGCTGCGTTCTCGTGAAATCCTCCTCGCGCAGCCAGCCCTGCTGGAACTGGAAGCGCCGATCAAGATCTGCGGTGACATCCATGGTCAGTACTACGATCTCCTCCGCCTCTTCGACAACGGTGGTTTCCCGCCGTCTGCGAATTACCTGTTCCTGGGTGACTACGTCGACCGCGGCAAGCAGGGTCTGGAGACCATCTGCCTGGTTCTGGCGTTCAAAGTTAAATTCCCGGAAAACTTCTTCCTGCTGCGTGGCAACCATGAATGCGCGTCTATCAACCGTATCTATGGTTTCTTCGATGAATGTAAACGTCGCTACAACATCCGCCTGTGGAAAGCGTTCACCGACACGTTCAACTGCCTGCCTGTTGCCTGCATTATCGACGACAAAATCTTTTGCTGCCACGGCGGTCTGAGCCCGGAACTGCAGACTATGGATCAGATCAAACGTATCGCGCGTCCGTGCGATGTTACCGATGCGGGTCTGATCTGTGACCTGCTGTGGTCTGACCCGGAGGAAGGTCTGTCTGGTTGGGGTGAAAACGACCGTGGCGTTTCTTATACCTTCGGTCAGGACATCGTTGAAAAATTCCTGCGTAAGCACGAGTTCGACCTGATCGTTCGTGCGCATCAGGTTGTTGAAGACGGTTACCAGTTCTTCGCAAAACGTCAGCTCATCACCATCTTCTCTGCGCCGAACTATTGCAACGAATTTGACAACTCTGGCGCGGTTATGTCTGTCGACGCGGACCTCCTGTGCTCTTTTCAGATCCTGAAACCGAGCGTGAAAAAACCGAAATTCTATCAGTAA

__ Codon Optimized Sequence: __

TACTTCCAATCC ATGAGCCTGGATGTTGAC GCTATCATCGACAAACTGCTGGGTGTTCGCGTTTCTAAACCGGGTAAACAGGTTAATCTGGCAGAAAACGATGTTAAAAACCTGGCTCTGCGTTCTCGTGAAATCCTGCTGGCGCAGCCAGCGCTCCTGGAACTGGAAGCGCCGATCAAGATCTGTGGTGACATTCACGGTCAATACTACGACCTGCTGCGTCTCTTCGACAATGGTGGCTTTCCGCCGTCCGCGAATTACCTGTTCCTGGGTGACTATGTAGACCGTGGTAAGCAAGGTCTGGAGACCATCTGCCTGGTTCTGGCCTTCAAAGTTAAATTCCCGGAAAACTTCTTCCTGCTCCGTGGCAATCACGAATGCGCGTCTATCAACCGTATCTACGGTTTCTTCGATGAATGCAAACGTCGTTACAACATTCGTCTCTGGAAAGCGTTCACCGACACTTTCAACTGCCTGCCGGTTGCGTGCATTATCGACGACAAAATCTTCTGCTGCCATGGTGGTCTCTCTCCGGAACTGCAGACCATGGACCAGATCAAACGTATCGCGCGTCCGTGCGACGTCACCGATGCGGGTCTGATCTGCGATCTGCTGTGGTCTGACCCGGAAGAGGGTCTGTCTGGTTGGGGTGAAAATGATCGTGGTGTTTCTTACACCTTCGGTCAGGACATCGTGGAAAAATTCCTGCGTAAACACGAGTTCGACCTGATCGTTCGTGCGCATCAGGTTGTTGAAGACGGCTACCAATTCTTTGCCAAACGCCAACTCATTACCATCTTCTCTGCGCCGAACTACTGCAACGAGTTTGACAACTCTGGTGCGGTTATGTCCGTTGATGCGGATCTCCTGTGCTCTTTCCAAATCCTCAAACCGTCTGTAAAGAAGCCGAAGTTCTACCAGTAACAGTAAAGGTGGATA

Query 6 CCTCGACGTTGACGCGATCATCGATAAACTGCTGGGTGTTCGTGTTTCTAAACCGGGTAA 65 ||| || |||||||| |||||||| ||||||||||||||||| ||||||||||||||||| Sbjct 6 CCTGGATGTTGACGCTATCATCGACAAACTGCTGGGTGTTCGCGTTTCTAAACCGGGTAA 65
 * ** Score ** || ** Expect ** || ** Identities ** || ** Gaps ** || ** Strand ** ||
 * 1234 bits(1368) || 0.0 || 818/907(90%) || 0/907(0%) || Plus/Plus ||
 * 1234 bits(1368) || 0.0 || 818/907(90%) || 0/907(0%) || Plus/Plus ||

Query 66 ACAAGTTAATCTGGCAGAAAATGACGTTAAAAACCTGGCGCTGCGTTCTCGTGAAATCCT 125 ||| ||||||||||||||||| || |||||||||||||| |||||||||||||||||||| Sbjct 66 ACAGGTTAATCTGGCAGAAAACGATGTTAAAAACCTGGCTCTGCGTTCTCGTGAAATCCT 125

Query 126 CCTCGCGCAGCCAGCCCTGCTGGAACTGGAAGCGCCGATCAAGATCTGCGGTGACATCCA 185 || ||||||||||| || ||||||||||||||||||||||||||||| |||||||| || Sbjct 126 GCTGGCGCAGCCAGCGCTCCTGGAACTGGAAGCGCCGATCAAGATCTGTGGTGACATTCA 185

Query 186 TGGTCAGTACTACGATCTCCTCCGCCTCTTCGACAACGGTGGTTTCCCGCCGTCTGCGAA 245 ||||| |||||||| || || || ||||||||||| ||||| || |||||||| ||||| Sbjct 186 CGGTCAATACTACGACCTGCTGCGTCTCTTCGACAATGGTGGCTTTCCGCCGTCCGCGAA 245

Query 246 TTACCTGTTCCTGGGTGACTACGTCGACCGCGGCAAGCAGGGTCTGGAGACCATCTGCCT 305 ||||||||||||||||||||| || ||||| || ||||| |||||||||||||||||||| Sbjct 246 TTACCTGTTCCTGGGTGACTATGTAGACCGTGGTAAGCAAGGTCTGGAGACCATCTGCCT 305

Query 306 GGTTCTGGCGTTCAAAGTTAAATTCCCGGAAAACTTCTTCCTGCTGCGTGGCAACCATGA 365 ||||||||| ||||||||||||||||||||||||||||||||||| |||||||| || || Sbjct 306 GGTTCTGGCCTTCAAAGTTAAATTCCCGGAAAACTTCTTCCTGCTCCGTGGCAATCACGA 365

Query 366 ATGCGCGTCTATCAACCGTATCTATGGTTTCTTCGATGAATGTAAACGTCGCTACAACAT 425 |||||||||||||||||||||||| ||||||||||||||||| |||||||| |||||||| Sbjct 366 ATGCGCGTCTATCAACCGTATCTACGGTTTCTTCGATGAATGCAAACGTCGTTACAACAT 425

Query 426 CCGCCTGTGGAAAGCGTTCACCGACACGTTCAACTGCCTGCCTGTTGCCTGCATTATCGA 485 || || |||||||||||||||||||| |||||||||||||| ||||| ||||||||||| Sbjct 426 TCGTCTCTGGAAAGCGTTCACCGACACTTTCAACTGCCTGCCGGTTGCGTGCATTATCGA 485

Query 486 CGACAAAATCTTTTGCTGCCACGGCGGTCTGAGCCCGGAACTGCAGACTATGGATCAGAT 545 |||||||||||| |||||||| || ||||| |||||||||||||| ||||| ||||| Sbjct 486 CGACAAAATCTTCTGCTGCCATGGTGGTCTCTCTCCGGAACTGCAGACCATGGACCAGAT 545

Query 546 CAAACGTATCGCGCGTCCGTGCGATGTTACCGATGCGGGTCTGATCTGTGACCTGCTGTG 605 |||||||||||||||||||||||| || |||||||||||||||||||| || |||||||| Sbjct 546 CAAACGTATCGCGCGTCCGTGCGACGTCACCGATGCGGGTCTGATCTGCGATCTGCTGTG 605

Query 606 GTCTGACCCGGAGGAAGGTCTGTCTGGTTGGGGTGAAAACGACCGTGGCGTTTCTTATAC 665 |||||||||||| || ||||||||||||||||||||||| || ||||| |||||||| || Sbjct 606 GTCTGACCCGGAAGAGGGTCTGTCTGGTTGGGGTGAAAATGATCGTGGTGTTTCTTACAC 665

Query 666 CTTCGGTCAGGACATCGTTGAAAAATTCCTGCGTAAGCACGAGTTCGACCTGATCGTTCG 725 |||||||||||||||||| ||||||||||||||||| ||||||||||||||||||||||| Sbjct 666 CTTCGGTCAGGACATCGTGGAAAAATTCCTGCGTAAACACGAGTTCGACCTGATCGTTCG 725

Query 726 TGCGCATCAGGTTGTTGAAGACGGTTACCAGTTCTTCGCAAAACGTCAGCTCATCACCAT 785 |||||||||||||||||||||||| ||||| ||||| || ||||| || ||||| ||||| Sbjct 726 TGCGCATCAGGTTGTTGAAGACGGCTACCAATTCTTTGCCAAACGCCAACTCATTACCAT 785

Query 786 CTTCTCTGCGCCGAACTATTGCAACGAATTTGACAACTCTGGCGCGGTTATGTCTGTCGA 845 |||||||||||||||||| |||||||| |||||||||||||| ||||||||||| || || Sbjct 786 CTTCTCTGCGCCGAACTACTGCAACGAGTTTGACAACTCTGGTGCGGTTATGTCCGTTGA 845

Query 846 CGCGGACCTCCTGTGCTCTTTTCAGATCCTGAAACCGAGCGTGAAAAAACCGAAATTCTA 905 ||||| |||||||||||||| || ||||| |||||| || || || ||||| ||||| Sbjct 846 TGCGGATCTCCTGTGCTCTTTCCAAATCCTCAAACCGTCTGTAAAGAAGCCGAAGTTCTA 905

Query 906 TCAGTAA 912 |||||| Sbjct 906 CCAGTAA 912

__ Forward Primer: __ 5’ _ TACTTCCAATCC ATGAGCCTGGATGTTGAC _ 3’ _30_ bp  GC Content _46.7%_% 0 mM Mg2+ Tm _62.0 __oC 1.5 mM Mg2+ Tm _69.0_ oC 2 mM Mg2+ Tm _69.5_ oC__ __ 4 mM Mg2+ Tm _70.5_ oC 6 mM Mg2+ Tm _71.0_ oC __

Reverse Primer __ : __ __ 5’ _ CCGAAGTTCTACCAGTAA TATCCACCTTTACTG _ 3’ ** Reverse complement it: **__ __ 5’ _ CCGAAGTTCTACCAGTAA CAGTAAAGGTGGATA _ 3’ _33_ bp __ GC Content _42.4_% __ 0 mM Mg2+ Tm _60.6 __ oC 1.5 mM Mg2+ Tm _67.9 __oC 2 mM Mg2+ Tm _68.5_ oC__ __ 4 mM Mg2+ Tm _69.5_ oC 6 mM Mg2+ Tm _69.6 __ oC

Complete pNIC28-Bsa4 with the gene inserted :

TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGCACCATCATCATCATCATTCTTCTGGTGTAGATCTGGGTACCGAGAACCTGTACTTCCAATCC ATG AGCCTGGATGTTGAC __<span style="background-color: #ffffff; font-family: Arial,sans-serif; font-size: 10pt;">GCTATCATCGACAAACTGCTGGGTGTTCGCGTTTCTAAACCGGGTAAACAGGTTAATCTGGCAGAAAACGATGTTAAAAACCTGGCTCTGCGTTCTCGTGAAATCCTGCTGGCGCAGCCAGCGCTCCTGGAACTGGAAGCGCCGATCAAGATCTGTGGTGACATTCACGGTCAATACTACGACCTGCTGCGTCTCTTCGACAATGGTGGCTTTCCGCCGTCCGCGAATTACCTGTTCCTGGGTGACTATGTAGACCGTGGTAAGCAAGGTCTGGAGACCATCTGCCTGGTTCTGGCCTTCAAAGTTAAATTCCCGGAAAACTTCTTCCTGCTCCGTGGCAATCACGAATGCGCGTCTATCAACCGTATCTACGGTTTCTTCGATGAATGCAAACGTCGTTACAACATTCGTCTCTGGAAAGCGTTCACCGACACTTTCAACTGCCTGCCGGTTGCGTGCATTATCGACGACAAAATCTTCTGCTGCCATGGTGGTCTCTCTCCGGAACTGCAGACCATGGACCAGATCAAACGTATCGCGCGTCCGTGCGACGTCACCGATGCGGGTCTGATCTGCGATCTGCTGTGGTCTGACCCGGAAGAGGGTCTGTCTGGTTGGGGTGAAAATGATCGTGGTGTTTCTTACACCTTCGGTCAGGACATCGTGGAAAAATTCCTGCGTAAACACGAGTTCGACCTGATCGTTCGTGCGCATCAGGTTGTTGAAGACGGCTACCAATTCTTTGCCAAACGCCAACTCATTACCATCTTCTCTGCGCCGAACTACTGCAACGAGTTTGACAACTCTGGTGCGGTTATGTCCGTTGATGCGGATCTCCTGTGCTCTTTCCAAATCCTCAAACCGTCTGTAAAGAAG __<span style="background-color: #00ffff; font-family: Arial,sans-serif; font-size: 10pt;">CCGAAGTTCTACCAG <span style="background-color: #ff0000; font-family: Arial,sans-serif; font-size: 10pt;">TAA <span style="background-color: #ff00ff; font-family: Arial,sans-serif; font-size: 10pt;">CAGTAAAGGTGGATA <span style="background-color: #ffffff; font-family: Arial,sans-serif; font-size: 10pt;">CGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATTGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGATCATGCGCACCCGTGGGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAAT

Figure 1: Gel with gene plus pNIC28-Bsa4



Figure 2: Gel with pNIC28-Bsa4 only



Figure 3: Gel with gene only


 * Restriction Enzyme Digest**

Objective: The purpose of this experiment was to digest pGBR22 plasmid with restriction enzymes and visualize fragments on a gel.

Figure 1: The RE Digest worked as planned. Lane 2 contains 5 uL of 1kg DNA ladder, Lane 3 contains 12 uL of uncut plasmid, Lane 4 contains 30 uL of sample one with EcoRI-HF, Lane 5 contains 30 uL of sample two with PvuII-HF, and Lane 6 contains 30 uL of sample three with both EcoRI-HF and PvuII-HF (buffer used was NEBuffer 4).



Figure 2: 1kb Ladder used in gel above.

Results/Conclusion: The bands appeared as expected. Lane 2 was the 1kb DNA Ladder. In Lane 3 the uncut plasmid is clearly visible and Lanes 4 through 6 all have 25 ul of samples 1, 2, or 3. The process of RE Digest is important in that it will have to repeated again with our target. RE Digest is especially important when we will want to cut the plasmid and find what can bind/fit into those cuts.


 * Week 1 & 2**
 * Aditi - good. So, this PCR was your only one? If so, redo this week to see if you can get it to work. - Dr. B 090913**


 * Analyzing DNA Sequencing **

Objective: The purpose of this lab was to BLAST sequences and compare sequences in order to determine the DNA sequence of a plasmid.

Figure 1: Screenshot of the lab from the Google Drive.

Results/Conclusions: In this experiment a primer sequence was used that was downloaded and altered in order to make a final reverse complement sequence with part of the original sequence in it. The final sequence was put into NEB where the primer was placed in the primer ring where a gel could be found. The gel was cut and the buffers for those enzymes were explored.


 * PCR and Agarose Gel Run **

Objective: The purpose of this experiment is to make a gel for analysis through making a master mix, also known as a coding sequence.

Figure One: Agarose gel with 1kb DNA ladder showing in the second lane. In lane three and seven sample A: a 1:1000 dilution template, lane four and eight sample B: a 1:1000 dilution differing in the amount of DDW, lane five and nine give sample C a 1:100 dilution and lane six and nine, sample D with no DNA (control sample). The picture was taken in the UV imaging box.

Analysis/Conclusions:

In this experiment four samples were made (A, B,C and D), each differing in the amount of DNA template, template dilution and diluted Taq, and were run in the PCR machine. Once finished in the machine the agarose gel was made and the samples were stained, inserted in the gel and ran. The results showed that the right amount of DNA was present and that the experiment was successful. Potential errors could have been made from incorrectly made dilutions, or temperature variances in certain solutions and the samples.


 * <span style="color: #222222; font-family: arial,sans-serif; line-height: 1.5;">PCR Primer Design for Primer Overlap Assembly PCR **

Objective: The purpose of this experiment was to design an oligo set of forward and reverse primers for PCR synthesizing and amplifying the CDS of the gene of interest so that it can be inserted into a cloning (or expression) vector.

| Helix Systems -- Center for Information Technology |

| http://helix.nih.gov |

| National Institutes of Health, Department of Health and Human Services |

| DNAWorks Web Site: http://helixweb.nih.gov/dnaworks |

Job started on 08/30/2013 at 16:14:02

Job name: AS083013Tcruzi

SEQUENCE 1: PROTEIN LENGTH = 304

1 MSLDVDAIIDKLLGVRVSKPGKQVNLAENDVKNLALRSREILLAQPALLELEAPIKICGD

61 IHGQYYDLLRLFDNGGFPPSANYLFLGDYVDRGKQGLETICLVLAFKVKFPENFFLLRGN

121 HECASINRIYGFFDECKRRYNIRLWKAFTDTFNCLPVACIIDDKIFCCHGGLSPELQTMD

181 QIKRIARPCDVTDAGLICDLLWSDPEEGLSGWGENDRGVSYTFGQDIVEKFLRKHEFDLI

241 VRAHQVVEDGYQFFAKRQLITIFSAPNYCNEFDNSGAVMSVDADLLCSFQILKPSVKKPK

301 FYQX

None found

24 oligonucleotides need to be synthesized

1 ATGAGCCTGGATGTTGACGC 20

2 CCCGGTTTAGAAACGCGAACACCCAGCAGTTTGTCGATGATAGCGTCAACATCCAGGCTC 60

3 TTCGCGTTTCTAAACCGGGTAAACAGGTTAATCTGGCAGAAAACGATGTTAAAAACCTGG 60

4 CTGGCTGCGCCAGCAGGATTTCACGAGAACGCAGAGCCAGGTTTTTAACATCGTTTTCTG 60

5 TGCTGGCGCAGCCAGCGCTCCTGGAACTGGAAGCGCCGATCAAGATCTGTGGTGACATTC 60

6 TTGTCGAAGAGACGCAGCAGGTCGTAGTATTGACCGTGAATGTCACCACAGATCTTGATC 60

7 GCTGCGTCTCTTCGACAATGGTGGCTTTCCGCCGTCCGCGAATTACCTGTTCCTGGGTGA 60

8 CAGATGGTCTCCAGACCTTGCTTACCACGGTCTACATAGTCACCCAGGAACAGGTAATTC 60

9 CAAGGTCTGGAGACCATCTGCCTGGTTCTGGCCTTCAAAGTTAAATTCCCGGAAAACTTC 60

10 ATAGACGCGCATTCGTGATTGCCACGGAGCAGGAAGAAGTTTTCCGGGAATTTAACTTTG 60

11 AATCACGAATGCGCGTCTATCAACCGTATCTACGGTTTCTTCGATGAATGCAAACGTCGT 60

12 TGAAAGTGTCGGTGAACGCTTTCCAGAGACGAATGTTGTAACGACGTTTGCATTCATCGA 60

13 GCGTTCACCGACACTTTCAACTGCCTGCCGGTTGCGTGCATTATCGACGACAAAATCTTC 60

14 TGGTCTGCAGTTCCGGAGAGAGACCACCATGGCAGCAGAAGATTTTGTCGTCGATAATGC 60

15 TCCGGAACTGCAGACCATGGACCAGATCAAACGTATCGCGCGTCCGTGCGACGTCACCGA 60

16 CCTCTTCCGGGTCAGACCACAGCAGATCGCAGATCAGACCCGCATCGGTGACGTCGCACG 60

17 GGTCTGACCCGGAAGAGGGTCTGTCTGGTTGGGGTGAAAATGATCGTGGTGTTTCTTACA 60

18 TACGCAGGAATTTTTCCACGATGTCCTGACCGAAGGTGTAAGAAACACCACGATCATTTT 60

19 TCGTGGAAAAATTCCTGCGTAAACACGAGTTCGACCTGATCGTTCGTGCGCATCAGGTTG 60

20 GTAATGAGTTGGCGTTTGGCAAAGAATTGGTAGCCGTCTTCAACAACCTGATGCGCACGA 60

21 GCCAAACGCCAACTCATTACCATCTTCTCTGCGCCGAACTACTGCAACGAGTTTGACAAC 60

22 GCACAGGAGATCCGCATCAACGGACATAACCGCACCAGAGTTGTCAAACTCGTTGCAGTA 60

23 GATGCGGATCTCCTGTGCTCTTTCCAAATCCTCAAACCGTCTGTAAAGAAGCCGAAGTTC 60

24 TTACTGGTAGAACTTCGGCTTCTTTACAGAC 31

FINAL SUMMARY FOR 1 SOLUTION


 * 1) Tm Len | Score TmRange Short Long #Olig #Repeat #Misprime

1 62 60 | 0.000 1.7 15 60 24 0 0

Results/Conclusion:

In this experiment oligoneleotides (primers) were found using the amino acid sequence for T. cruzi serine/threonine protein phosphatase catalytic subunit, putative. These primers were ordered for further experiments regarding this target. Errors could have occurred from the use of the information that was previously provided on the VDS Targets page.


 * Nanodrop Spectrophotometer: Determine Concentration of Plasmids, DNA; Submitting DNA to DNA Sequencing Facility **

Objective: The purpose of this lab was to prepare a for the core lab for them to add a primer to the solution for later analysis.

Option A from the protocol was chosen.



Figure 1: Documented proof that a request was sent using core website requesting for in-house primer M13F to be add and DNA sequencing to be done on the prepared template of DNA (pGBR22).

Results/Conclusion:

In this experiment, with the use of pGBR22, a DNA template was made and taken to a different lab after the proper request form was sent in. The primer chosen was M13F and the template was taken to the lab the next day after making it in the wet lab. The results were emailed several hours later and the analysis of those results took place in the Analyzing DNA Sequence lab. Errors also could have been caused in the lab as well if the pGBR22 was not kept at the right temperature during preparation.


 * Nanodrop Spectrophotometer: Determine Concentration of Plasmids, DNA; Quantifying DNA using Nanodrop **

Objective: The purpose of this experiment was to refresh our memory on how to use the nanodrop spectrophotometer for future experiments and to take measurements of pGBR22 properly.



Figure 1: Plot of pGBR22 (205.4 ng/uL) using the nanodrop spectrophotometer that found the concentration to be 173.8 ng/uL.



Figure 2: Plot of pGBR22 (205.4 ng/uL) using the nanodrop spectrophotometer that found the concentration to be 163.4 ng/uL.

Results/Conclusion: In this experiment, how to properly take measurements utilizing the nanodrop spectrophotometer was relearned. pGBR22's original concentration is 205.4 ng/uL. The concentration was found to be 173.8 ng/uL in the first measurement with a 260/280 value of 1.86 and a 260/230 value of 2.05, which is close to the original concentration value. The second measurement found the concentration to be 163.4 ng/uL with a 260/280 value of 1.85 and a 260/230 value to be 1.94. The average of the two concentrations measured was found to be 168.6 ng/uL, which is not very close to the actual value. Errors could have been made during the experiment if the machine was not properly blanked skewing future data or some of the water may not have been wiped off properly.