Jessica+N.

Week 15&16 Objective: The purpose of this lab is to purify the protein using the affinity tag and Ni-NTA resin.
 * Protein Purification**

Objective: The purpose of this lab is to make proteins for enzyme assays.
 * Protein Expression**

Analysis: After growing the bacterial culture overnight, the OD600 of the culture was measured using the Vernier Visible Spectrum (Chipper). At 600nm, the OD600 reached 0.496. IPTG was added and the proceed to the induction step. The pellet was harvested by centrifugation and weight approximately 2.44g. After sanitation, the sample was spun down to remove the soluble fraction for purification.

Week 13&14
 * Protein Expression**

Objective: The purpose of this procedure is to prepare PCR inserts and accepting vector for cloning.
 * Cloning - Cohesive End Generation, Annealing & Transformation**

Figure 2. Plate A of H. pylori of alpha carbonic anhydrase with pNIC-Bsa4 on an LB+KAN+SUC plate from Tube A samples: 1 microliters of PCR insert and 7 microliters of accepting vector.

Analysis: The cohesive end generation on PCR inserts and on the accepting vector were done in tandem in order to prepare the samples for the next step - annealing and transformation. Two plates from made each from Tube A and Tube B, containing the different amounts of PCR inserts and the accepting vector. Both plates showed no colonies. The next step will be to move on to the surrogate and start protein expression.

Great work. Did you submit to sequencing? -UM Week 11&12 Objective: The purpose of this lab is to prepare the plasmid as an accepting vector by cutting with the restriction enzyme BsaI-HF in order to proceed to cohesive end generation. Analysis: The concentrations for pNIC-Bsa4 as the accepting vector were 66.3 ng/uL and 68.0 ng/uL, respectively. The next step would be to perform cohesive end generation with the plasmid and PCR product.
 * Cloning - Preparation of pNIC-Bsa4 as Accepting Vector**

Objective: The purpose of this protocol is to extract the DNA from transformed bacterial cells. Analysis: After the plasmid DNA was extracted and purified using Midi prep, the concentration was taken and came out to be 115.5 ng/uL. The 260/280 was 1.90, and the 260/230 was 2.79. The next step will be to prepare for cloning by preparing the pNIC-Bsa4 as accepting vector.
 * Plasmid DNA Purification by Midi Prep Protocol**

Objective: The purpose of this lab is to transform the bacterial cells with plasmid DNA to make more plasmid DNA. Figure 1. Pellets containing E.coli DH5α + pNIC-Bsa4.
 * Transformation of Competent Cells for Plasmid Prep of pNIC-Bsa4**

Analysis: Bacterial colonies were put in LB media, placed in the 37 degrees C shaking incubator overnight, and centrifuged the next day in order to retain the pellet. Next step was to purify the plasmid DNA by Midi prep.

Objective: Made more PCR Cleanup for second trial of cloning. Primary PCR -> Secondary PCR -> PCR^2 -> PCR Cleanup Analysis: Because the first trial of cloning failed, more PCR product was needed in order to proceed to the second trial of getting a positive clone. After PCR Cleanup, the average concentration was measured and came out to be 197.3 ng/uL. The 260/280 was 1.83 and 1.81, respectively. The 260/230 was 2.17 and 2.06, respectively. The next step will be to transform the bacteria with plasmid DNA.
 * Making More PCR Product**


 * DNA Sequencing**

Analysis: After getting the results back, BLAST all of the results against the original DNA sequence. However, there was one mismatch in the beginning of the sequence, therefore, there was no positive clone.

Very good! Looks like you got a lot done in lab the past two weeks. Hope you get a positive clone. -UM Week 9 & 10 Objective: The purpose of Miniprep is to extract and purify the plasmid DNA from the cell. Nanodrop was performed to determine the concentrations of the DNA. Analysis: All eight tubes contained pellets, but the three pellets (Figures 1-3) showed the higher concentrations. Sample 7 from Tube B had the highest concentration among the eight tubes with a 260/280 of 1.83 and 260/230 of 1.63. Possibly low concentration may be the result of adding 100 microliters of the elution solution instead of 50 microliters.
 * Miniprep & Nanodrop**

Objective: The purpose of making the master plate is to conglomerate the PCR inserts and accepting vector to get positive matches from DNA sequencing.
 * Master Plate**

Analysis: 1-8 all had one colony from overnight in the incubator. When all eight tubes were spun down, all of the tubes formed a pellet.

Objective: The purpose of this procedure is to prepare PCR inserts and accepting vector for cloning.
 * pNIC-Bsa4 Cloning - Cohesive End Generation, Annealing & Transformation**

Analysis: The cohesive end generation on PCR inserts and on the accepting vector were done in tandem in order to prepare the samples for the next step - annealing and transformation. Two plates from made each from Tube A and Tube B, both containing the same amount of PCR inserts, but different amounts of the accepting vector. Plate A showed more colonies than Plate B.


 * Virtual Screening - Creating Homology Model**

Amino Acid Sequence of Target: MKKTFLIALALTASLIGAENTKWDYKNKENGPHRWDKLHKDFEVCKSGK SQSPINIEHYYHTQDKADLQFKYAASKPKAVFFTHHTLKASFEPTNHIN YRGHDYVLDNVHFHAPMEFLINNKTRPLSAHFVHKDAKGRLLVLAIGFE EGKENPNLDPILESVQKKQNFKEVALDAFLPKSINYYHFNGSLTAPPCT EGVAWFVIEEPLEVSAKQLAEIKKRMKNSPNQRPVQPDYNTVIIKSSAETR

Top Template PDB ID: 4g7aB Identities=39% Positives=55%

__ Template Molprobity Scores __ Clashscore=7.73 Molprobity score=1.69

__Homology Molprobity Scores__ Clashscore=7.16 Molprobity score=1.66

Objective: The purpose of this experiment was to transfer the gene of interest into the protein expression vector pNIC-Bsa4. Figure 1. The concentration of the plasmid after PCR cleanup to prepare the DNA as an accepting vector.
 * pNIC-Bsa4 Cloning - Preparation of pNIC-Bsa4 as Accepting Vector**

Analysis: After Midiprep, pNIC-Bsa4 was prepared to be used as the accepting vector for cloning. The concentration was high enough to proceed to the next step of cloning.

Objective: The purpose of this lab was to extract the DNA from transformed bacterial cells in order to obtain just the plasmid. Trial 4: 10/25/13 Figure 1. Concentration of the eluted DNA after Midiprep.
 * Re-do of Midiprep**

Analysis: After four trials of growing pNIC-Bsa4 and performing Midiprep, the concentration of the final eluted DNA was high enough to proceed to the next step.

Nice work. Could use a little more analysis. Try the pNIC again, the concentrations looked a little low. -UM Week 7 & 8 Objective: The purpose of this was to transform the E. coli DN5α with the plasmid pBIC-Bsa4 to make more plasmid DNA Tube 1-4: pellet containing E.coli DH5α + pNIC-Bsa4 and LB+KAN - 10 microliters
 * Re-do of Transforming Bacterial Cells**

Analysis: One colony that grew from each plate were placed in LB media and Kanamycin stock was added and was placed in the 37 degree shaker overnight for approximately 16 hour. The next day, in the morning, the liquids were centrifuged and the bacteria pellet was placed in the -20 degree freezer.

Objective: The purpose of this experiment was to transfer the gene of interest into the protein expression vector pNIC-Bsa4.
 * pNIC-Bsa4 Cloning - Preparation of pNIC-Bsa4 as Accepting Vector**

Trial 1: 10/16/13 The concentration was 2.8 ng/ul, which is a very low concentration.

Trial 2: 10/17/13 The concentration came out to be 2.1 ng/ul.

Trial 3: 10/18/13 The concentration came out to be 8.3 ng/ul.

Analysis: To determine if pNIC-Bsa4 could be used as an accepting vector for the gene of interest, PCR clean up was performed to see if the concentration was high enough to transfer the gene into the vector. In the first two trials, the incorrect wash solution was used - ethanol was not contained in the bottle - so the concentration was very low. In the third trial, it was assumed that a higher concentration would be obtained by using the correct ethanol-containing wash solution, however, the concentration was low again - 8.3 ng/ul. Thus, the bacteria culture had to be grown again.

Objective: The purpose of this lab was to extract the DNA from transformed bacterial cells in order to obtain just the plasmid. Analysis: By using the Midi Prep kit, the DNA from the bacterial cells were extracted. After using several necessary buffers inside the kit, the cell was lysed, releasing every component except the plasmid, such the cell wall, cell membrane, proteins and other inclusions. The concentration came out to be 23.5 ng/ul, the 260/280 value was 2.02, and the 260/230 value was -1.88. By looking at the purity values, it can be determined that the DNA was contaminated from other contaminants.
 * MidiPrep**

Objective: The purpose of this lab was to determine the concentration of the DNA by using spectrophotometry. Analysis: The concentration of the final DNA product from using nanodrop came out to be 67.3 ng/ul, which is a pretty high concentration.At wavelength of 230nm, the absorbance was 0.635. The purity was also determined by the 260/280 and 260/230 values, which tells how the pure the DNA is in relation to the protein, and in the presence of other contaminants, respectively. The 260/280 value was 1.80 and 260/230 was 2.12, which tells the the DNA is relatively pure.
 * PCR Cleanup & Nanodrop**

Great work! be sure to include a ladder image. Great job on the progress! - Michael T. Week 5 & 6 Objective: The purpose of this was to transform the E. coli DN5α with the plasmid pBIC-Bsa4 to make more plasmid DNA.
 * Transformation of Competent Cells for Plasmid Prep of pNIC-Bsa4**

(starting from left) Tube 1 & 2: pellet containing E.coli DH5α + pNIC-Bsa4 and LB+KAN - 10 microliters Tube 3 & 4: pellet containing E.coli DH5α + pNIC-Bsa4 and LB+KAN - 50 microliters

Analysis: The procedure took three days to perform. On the first day, the bacteria was added to LB media and was incubated overnight. The next day, in the afternoon, one colony that grew from each plate were placed in LB media and Kanamycin stock was added and was placed in the 37 degree shaker overnight for approximately 16 hour. The third day, in the morning, the liquids were centrifuged and the bacteria pellet was placed in the -20 degree freezer.

Objective: The purpose of this lab was to make LB media and LB plates to perform the transformation step.
 * LB Media and LB plate**

Analysis: The LB media was made by adding Bacto-tryptone, Bacto-yeast extract, and NaCl, and autoclaving. The LB plates were made by adding the same compounds but, in addition, including the antibiotic, Kanamycin stock. This lab was performed for the next step - transformation of competent cells for plasmid preparation of pNIC-Bsa4.

Objective: The purpose of this lab was to produce lots of copies of the PCR products because most of them will be lost during the PCR cleanup. Lane 1: 1kb DNA ladder Lanes 2-4: PCR Squared reactions (contain 5X rxn buffer, dNTP, secondary PCR reaction, Forward and Reverse primers, Q5, and nanopure water)
 * PCR Primer Overlap - PCR Squared**

Analysis: The PCR squared reactions which amplified the reaction is shown on the 1% agarose gel. The 100bp DNA ladder was not in the -20 degrees freezer, so instead the 1kb DNA ladder was used. However, the size of the gene came out to be around 741 nt. Many copies were made because a lot of the products will eventually be lost in PCR cleanup.

Objective: The purpose of this experiment was to insert the forward and reverse primers into the primary PCR reaction to piece them together. Lane 1: Empty Lane 2: 100 bp DNA ladder Lane 3: Secondary PCR reaction.
 * PCR Primer Overlap - Secondary PCR**

Analysis: Lane 3, according to the image, showed the secondary PCR reaction with a size of the gene was about 741 nt, which is similar to the size of the gene from the output file.

Week 3 & 4 Jessica - good work - include a ladder image - Dr. B 092713

Objective: The purpose of this experiment was to fill in the gaps between the oligos and piece together a full length DNA for the gene by conducting PCR.
 * PCR Primer Overlap - Primary PCR**

Lane 1: Empty Lane 2: 100 bp DNA ladder Lane 3: Primary PCR reactions on 1% agarose gel.

Analysis: The Lane 3 shows the overlapping of the DNA with the nucleotides filling in the gaps in the oligo mix. The gel needs to run longer so the reaction comes down and appears as a more smeared image.

__Ordered Forward and Reverse Primers of Gene:__

Objective: The purpose of this lab was to prepare an oligo mix in order to synthesize the gene of interest in future experiments.
 * PCR Primer Overlap - Oligo Mix**

Gene: HP Alpha CA

Analysis: The primers that were ordered were put together to create an oligo mix for future experimentation. In the later steps, PCR will be utilized to fill in the gaps between the oligos and piece together a full length of DNA for the gene.

Objective: The purpose of this lab was to design a forward and reverse primer from the gene (Alpha Carbonate Dehydratase) sequence obtained from the CDS and to synthesize compatible ends for LIC. After designing the primers, the gene would be inserted into the pNIC-Bsa4 as the accepting vector and cut with enzyme BsaI for further expression
 * PCR Primer Design Tails for pNIC-Bsa4 Cloning**

0.4 uM Oligo concentration 50 mM Na+ 0.3 mM dNTPs 0 mM Mg++

5’ TACTTCCAATCCATGAAAAAAACCTTCCTG 3’ 30 bp GC Content=36.7% 0mM Mg2+ Tm=58.1°C 1.5mM Mg2+ Tm=65.8ºC __ 2mM Mg2+ Tm=66.4ºC __ 4mM Mg2+ Tm=67.5ºC 6mM Mg2+ Tm=68.1ºC
 * Forward Primer: **

5’ TATCCACCTTTACTGTTAACGGGTCTTTGCAGA 3’ 33 bp GC Content=42.4% 0mM Mg2+ Tm=62.4°C 1.5mM Mg2+ Tm=69.8ºC __ 2mM Mg2+ Tm=70.3ºC __ 4mM Mg2+ Tm=71.3ºC 6mM Mg2+ Tm=71.8ºC
 * Reverse Primer: **

pNIC-Bsa4 Sequence:







Analysis: The coding sequence for Alpha Carbonate Dehydratase was used along with both upstream and downstream primers to perform LIC cloning. The sequence was then inserted in the pNIC-Bsa4 and was cut with enzyme BsaI, giving two restriction sites. The forward primer has a GC content of 36.7% with 30bp. The reverse primer had a GC content of 42.4% with 33bp and had a much higher melting point of 70.3 degrees Celcius than that of the forward primer.

Objective: The purpose of this lab was to digest the pGBR22 plasmid with certain restriction enzymes - EcoRI, PvuII, and both, and to visualize the restriction sites on the agarose gel.
 * Restriction Enzyme Digest**

Lane 1: Empty. Lane 2: 1kb DNA ladder. Lane 3: Uncut plasmid containing diluted plasmid and nanopure water. Lane 4: pGBR22 plasmid cut with EcoRI. Lane 5: pGBR22 plasmid cut with PvuII. Lane 6: pGBR22 plasmid cut with EcoRI + PvuII.

Analysis: Compared to the 1kb DNA ladder, there seemed to be no plasmid present for the uncut plasmid. Lane 4, plasmid cut with EcoRI, showed the plasmid at 4.0kb with one restriction site. Lane 5, cut with enzyme PvuII, showed the plasmid at 3.0kb and 1.0kb - two restriction enzymes. Lane 6, cut with EcoRI and PvuII, showed the plasmid at three restriction sites - 3.0kb, slightly below 1.0kb and slightly below 0.5kb. The intensity of the UV light appeared strongest where the DNA was most present.

Lane 1:Empty. Lane 2: 100bp DNA ladder. Lane 3: Tube A consisting 1:1000 dilution template with 18 uL of nanopure water; 0.3ng of plasmid. Lane 4: Tube B consisting 1:1000 dilution template with 9 uL of nanopure water; 3ng of plasmid. Lane 5: Tube C consisting 1:100 dilution template with 9 uL of nanopure water; 30ng of plasmid. Lane 6: Tube D consisting of no DNA template.
 * PCR Trial 2**

Analysis: The size of the DNA is around 1000bp. The intensity of the UV light is stronger in lanes B and C because more DNA plasmid is present in those locations. The pGBR22 also consisted of M13 Forward and M13 Reverse primers.

Week 1 & 2


 * PCR Primer Design for Primer Overlap Assembly PCR**

Objective: The purpose of the experiment was to design a set of oligo primers of both reverse and forward for further experimentation.

Figure 1. Logfile of Helicobacter pylori ACD.

Protein length: 416 Total size of gene: 1248 nt

DNA Sequence: 1 ATGAAAAAAACCTTTCTGATCGCGCTCGTACTCGCGACTTCTCTCATCGGTGCGGAAAAC 61 GCGAAGTGGGATTACAAAAACAAAGAGAACGGTCCGCATCGTTGGGATAAGCTGCACAAA 121 GACTTCGAGGTATGCAAATCTGGTAAGTCTCAGTCTCCGATCAACATCGAGCACTATTAC 181 CATACCCAGGACAAAGCGGATCTGCAATTTAAGTACGCGGCGTCTAAACCTAAAGCAGTT 241 TTCTTCACCCACCACACCCTCAAAGCATCTTTCGAACCGACCAACCACATCAATTACCGT 301 GGTCACGACTACGTTCTGGACAACGTTCACTTCCACGCGCCGATGGAGTTCCTCATCAAC 361 AACAAGACCCGTCCTCTCAGCGCGCACTTCGTTCACAAGGACGCGAAAGGTCGTCTGCTG 421 GTACTGGCGATCGGTTTCGAAGAAGGTAAAGAAAATCCGAACCTCGATCCGATCCTGGAA 481 GGTATCCAGAAAAAACAGAACTTCAAAGAAGTTGCGCTCGACGCGTTCCTGCCGAAATCC 541 ATCAACTACTACCACTTCAACGGTTCTCTGACTGCCCCTCCGTGCACCGAAGGTGTTGCG 601 TGGTTTGTTATCGAGGAACCGCTGGAAGTTTCCGCCAAGCAGCTGGCGGAGATCAAAAAA 661 CGTATGAAGAACTCTCCAAACCAGCGTCCGGTTCAGCCGGACTACAACACTGTTATCATC 721 AAATCTTCTGCGAAAACTCGTCTCGGCGACTCCCCAGGTTACGTTATGTCTAATATCGAA 781 TTCCGTCAACTCACTCGCGGTCACTCTCCGTCTGACGAACGTGAAGCGCGTCGTGTAGAA 841 GAGGCGGGTGGTCAGCTGTTCGTTATCGGTGGTGAACTCCGCGTCAACGGCGTTCTGAAC 901 CTGACCCGTGCTCTGGGCGACGTGCCGGGTCGTCCGATGATCTCTAATGAACCAGAAACC 961 TGTCAGGTGCCTATTGAATCTTCCGACTACCTGGTTCTGCTCGCTTGCGACGGCATTTCT 1021 GACGTGTTCAACGAACGCGACCTGTACCAACTGGTTGAAGCCTTTGCGAATGACTACCCG 1081 GTGGAAGACTACGCGGAACTGTCTCGTTTCATCTGCACGAAAGCGATTGAAGCGGGTAGC 1141 GCGGATAACGTTTCTGTCGTCATTGGTTTCCTGCGTCCGCCGCAAGACGTTTGGAAACTG 1201 ATGAAGCACGAATCCGACGACGAAGATAGCGACGTCACCGACGAGGAG

Oligotide Sequence: 1 ATGAAAAAAACCTTTCTGATCGCGCTCGTACTCGCGACTTCTCTCATCGGTGCGGAA 57 2 GATGCGGACCGTTCTCTTTGTTTTTGTAATCCCACTTCGCGTTTTCCGCACCGATGAGAGA 61 3 AAAGAGAACGGTCCGCATCGTTGGGATAAGCTGCACAAAGACTTCGAGGTATGCAAATCTG 61 4 GGTAATAGTGCTCGATGTTGATCGGAGACTGAGACTTACCAGATTTGCATACCTCGAAGTC 61 5 GATCAACATCGAGCACTATTACCATACCCAGGACAAAGCGGATCTGCAATTTAAGTACGCG 61 6 GGTGTGGTGGGTGAAGAAAACTGCTTTAGGTTTAGACGCCGCGTACTTAAATTGCAGATCC 61 7 TTTTCTTCACCCACCACACCCTCAAAGCATCTTTCGAACCGACCAACCACATCAATTACCG 61 8 CGCGTGGAAGTGAACGTTGTCCAGAACGTAGTCGTGACCACGGTAATTGATGTGGTTGGTC 61 9 ACGTTCACTTCCACGCGCCGATGGAGTTCCTCATCAACAACAAGACCCGTCCTCTCAGCGC 61 10 GCCAGTACCAGCAGACGACCTTTCGCGTCCTTGTGAACGAAGTGCGCGCTGAGAGGACGGG 61 11 TCGTCTGCTGGTACTGGCGATCGGTTTCGAAGAAGGTAAAGAAAATCCGAACCTCGATCCG 61 12 ACTTCTTTGAAGTTCTGTTTTTTCTGGATACCTTCCAGGATCGGATCGAGGTTCGGATTTT 61 13 AGAAAAAACAGAACTTCAAAGAAGTTGCGCTCGACGCGTTCCTGCCGAAATCCATCAACTA 61 14 GGTGCACGGAGGGGCAGTCAGAGAACCGTTGAAGTGGTAGTAGTTGATGGATTTCGGCAGG 61 15 GCCCCTCCGTGCACCGAAGGTGTTGCGTGGTTTGTTATCGAGGAACCGCTGGAAGTTTCCG 61 16 GGAGAGTTCTTCATACGTTTTTTGATCTCCGCCAGCTGCTTGGCGGAAACTTCCAGCGGTT 61 17 TCAAAAAACGTATGAAGAACTCTCCAAACCAGCGTCCGGTTCAGCCGGACTACAACACTGT 61 18 GGGGAGTCGCCGAGACGAGTTTTCGCAGAAGATTTGATGATAACAGTGTTGTAGTCCGGCT 61 19 GTCTCGGCGACTCCCCAGGTTACGTTATGTCTAATATCGAATTCCGTCAACTCACTCGCGG 61 20 CCTCTTCTACACGACGCGCTTCACGTTCGTCAGACGGAGAGTGACCGCGAGTGAGTTGACG 61 21 CGCGTCGTGTAGAAGAGGCGGGTGGTCAGCTGTTCGTTATCGGTGGTGAACTCCGCGTCAA 61 22 ACCCGGCACGTCGCCCAGAGCACGGGTCAGGTTCAGAACGCCGTTGACGCGGAGTTCACCA 61 23 GCGACGTGCCGGGTCGTCCGATGATCTCTAATGAACCAGAAACCTGTCAGGTGCCTATTGA 61 24 AAATGCCGTCGCAAGCGAGCAGAACCAGGTAGTCGGAAGATTCAATAGGCACCTGACAGGT 61 25 CGCTTGCGACGGCATTTCTGACGTGTTCAACGAACGCGACCTGTACCAACTGGTTGAAGCC 61 26 GACAGTTCCGCGTAGTCTTCCACCGGGTAGTCATTCGCAAAGGCTTCAACCAGTTGGTACA 61 27 GAAGACTACGCGGAACTGTCTCGTTTCATCTGCACGAAAGCGATTGAAGCGGGTAGCGCGG 61 28 ACGTCTTGCGGCGGACGCAGGAAACCAATGACGACAGAAACGTTATCCGCGCTACCCGCTT 61 29 GTCCGCCGCAAGACGTTTGGAAACTGATGAAGCACGAATCCGACGACGAAGATAGCGACGT 61 30 CTCCTCGTCGGTGACGTCGCTATCTTCGTCGT 32


 * Quantifying DNA Using Nanodrop**

Objective: The purpose of this experiment was to determine the concentration and absorbance of the DNA plasmid.





Results/Conclusion: The average concentration of the protein pGBR22 came out to be 397.6 ng/uL, which is pretty close to the originally obtained concentration of 365 ng/uL. Also, the purity of the sample in relation to protein (260/280) came out to an average of 1.88 and the purity in relation to other contaminants (260/230) came out to an average of 2.50. Thus, the protein is relatively pure.


 * Submit to DNA Sequencing**

__pGBR22 Forward Primer Sequence: __ NNNNNNNNNNNNNGGNGANTGGGCCCGACGTCGCATGCTCCCGGCCGCCATGGCCGCGGGNTTTTAGTGATGGTGATGGTGATGACCGAGCAAAGAGTGGCGTGCAATGGATATTTCACACTGCTCAACAAATGTGTAATCCTTGTTGTGACTGGTTACATCCAGTTTGCGGTCAACATAGTGATACCCTGGCATCCTCACAGGCTTCTTTGCCTTGTAAGTAGATTTGAATTCACACAAATAGTAACCACCTCCTTCCAACTTCAGAGCCATAAAGTTGTTTCCTATCAGCATTCCATCTCGTGCAAAGAGACGCTCAGTGTTGGGTTCCCAGCCCTGTGTCTTCTTCTGCATAACAGGTCCATTGGGAGGAAAGTTCACACCAGAGATTTTGACATTGTAGATGAAACAGTTGCCTTGGATGCTGGAATCATTGCTGACAGTACACACTGCACCATCTTCAAAGTTCATGATCCTCTCCCATGTATATCCCTCAGGGAATGACTGCTTTACATAATCAGGGATGTCTTCAGGGTACTTGGTGAATGGTATGCTTCCGTATTGAGACAGTGGTGATAAAATATCCCAAGCAAATGGCAGAGGTCCACCCTTGGTGACAGTGAGCTTTACCGTCTGCTCCCCCTCGTAAGGCTTTCCTTTTCCATCGCCTTCGACCTCAAAGTAGTGTCCATTGACCGTGCCTGACATATAAACCTTGTAGGTCATTTGTTTAGCGATCACACTCATGATATTTCTCCTTCAATCAATCAAAATCACTAGTGCGGCCGCCTGCAGGTCGACCATATGGGAGAGCTCCCAACGCGTTGGATGCATAGCTTGAGTATTCTATAGTGTCACCTAAATAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTNGNAAANCTGTCNTGCCAGCTGCNTNATGAATCGGCNANGCGCGGGGANANGCNGNTNGCGTATTGGGCGCTCNTCNNTTCCTCNNTCANTGACTCNNNNCNCTNGNCNTNNNNNNNNNNNNNGNNTCANCTCNNTNNANGNGNANTANNNNNNNNNNNNNNCNNGNNNANNNNNNANNANNNNNNNNNNNNNNNNNNNNNNNNNNNNNAAAANNNCNNNNNNNNNNNNNNTTTTNNNNNNNNNNNN

__pGBR22 Reverse Primer Sequence __

NNNNNNNNNNNNNNNGANNATAGAATACTCAAGCTATGCATCCAACGCGTTGGGAGCTCTCCCATATGGTCGACCTGCAGGCGGCCGCACTAGTGATTTTGATTGATTGAAGGAGAAATATCATGAGTGTGATCGCTAAACAAATGACCTACAAGGTTTATATGTCAGGCACGGTCAATGGACACTACTTTGAGGTCGAAGGCGATGGAAAAGGAAAGCCTTACGAGGGGGAGCAGACGGTAAAGCTCACTGTCACCAAGGGTGGACCTCTGCCATTTGCTTGGGATATTTTATCACCACTGTCTCAATACGGAAGCATACCATTCACCAAGTACCCTGAAGACATCCCTGATTATGTAAAGCAGTCATTCCCTGAGGGATATACATGGGAGAGGATCATGAACTTTGAAGATGGTGCAGTGTGTACTGTCAGCAATGATTCCAGCATCCAAGGCAACTGTTTCATCTACAATGTCAAAATCTCTGGTGTGAACTTTCCTCCCAATGGACCTGTTATGCAGAAGAAGACACAGGGCTGGGAACCCAACACTGAGCGTCTCTTTGCACGAGATGGAATGCTGATAGGAAACAACTTTATGGCTCTGAAGTTGGAAGGAGGTGGTTACTATTTGTGTGAATTCAAATCTACTTACAAGGCAAAGAAGCCTGTGAGGATGCCAGGGTATCACTATGTTGACCGCAAACTGGATGTAACCAGTCACAACAAGGATTACACATTTGTTGAGCAGTGTGAAATATCCATTGCACGCCACTCTTTGCTCGGTCATCACCATCACCATCACTAAAATCCCGCGGCCATGGCGGCCGGGAGCATGCGACGTCGGGCCCAATTCGCCCTATAGTGAGTCGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAANANGCCCGCACCGATCGCCCNTNCCCANCAGTNNNNCAGCCTGNATGGCNANGGNNGCNNCCTGNANNGNGCATTANNNCGGNGGGNGNNGNNGNNCGCNNANCGNGACNNTANNNNNNNGCNCCNNANNNNNNNCNTNNGNTTNNNNNNNNTTNNCNNNNNNTNNNNNTTNCCNNNAGNNNTAANNNGGGNTNNNTNNNNNNNNATTNNNGNNNNNNNNNNACNNNNNNNNNNNGNNNNNNGGNNNNNNNNNNNNNNNNNNNNNNNNCCNNNN


 * PCR**

Objective: The purpose of this lab was to, by using the Forward and Reverse Primers, intensify the coding sequence of the purple protein, and also to test four different samples by diluting the DNA primer. Figure1. Agarose gel consisting the gel ladder and four different samples dilutions of DNA primer. Lane 1: Empty Lane 2: 100 bp ladder Lane 3: Tube A consisting 1:1000 dilution template with 18 uL of nanopure water. Lane 4: Tube B consisting 1:1000 dilution template with 9 uL of nanopure water. Lane 5: Tube C consisting 1:100 dilution template with 9 uL of nanopure water. Lane 6: Tube D consisting of no DNA template.

Analysis/Conclusion: After running the gel, the agarose gel went through the UV imaging box. By comparing to the ladder, the diluted primers seem to be the size of 100bp. However, the DNA may not have been diluted correctly because there are so much DNA present on top.


 * Analyzing DNA Sequencing**

Objective: The purpose of this lab was to analyze and determine the DNA sequence of the plasmid by using BLAST.