ResearchPage+-+Johnny

Johnny's Research

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No progress was made due to poor time management. Hours spent in lab were in efforts to bolster //Salmonella// Research Report. The following week will consist of a goal to reach 30 hours of lab time, and to finish Week 6 and Week 7 progress.=====

Week 7


//Agarose Gel Information:// The primers used to to synthesize the gene of Carbonic Anyhdrase - Salmonella (PDB #3QY1) were E1-D14 for a total of 14 primers. Similar KOD PCR reagents in each PCR well included 10x rxn buffer, 25mM MgSO4, 2 mM dNTPs, dH20, and most importantly, KOD hotstart Polymerase (which must be kept cold). As seen, within the 7th well a smear appears in addition to a slight band in the 8th well. Although not as accurate of a gel as desired, it is cause to proceed on to PCR squared.
 * Lane 1: SKIPPED
 * Lane 2: SKIPPED
 * Lane 3: SKIPPED
 * Lane 4: SKIPPED
 * Lane 5: SKIPPED
 * Lane 6: 100 bp ladder
 * Lane 7: 1' (Primary) PCR mix
 * Lane 8: 2' (Secondary) PCR mix

>gi|16763390:c202072-201410 Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 chromosome, complete genome: ATGAAAGACATAGATACACTCATCAGCAACAATGCACTATGGTCAAAAATGCTGGTGGAAGAGGACCCCGGATTTTTTGAGAAACTGGCGCAAG CGCAAAAACCGCGCTTTCTATGGATTGGATGTTCCGACAGCCGCGTTCCCGCAGAGCGTTTAACCGGGCTTGAACCGGGCGAATTGTTTGTTCA CCGTAATGTGGCTAACCTGGTTATTCACACCGATCTGAACTGTCTCTCCGTGGTTCAGTATGCGGTAGATGTTCTGGAAGTTGAGCATATTATCATT TGCGGCCACTCCGGTTGCGGCGGTATCAAGGCTGCGGTAGAAAACCCTGAGCTGGGCTTGATTAATAACTGGCTGCTGCATATCCGCGACATCTG GCTTAAACATAGCTCGCTGTTGGGAAAAATGCCCGAAGAGCAACGTCTGGACGCGCTCTATGAATTGAACGTCATGGAACAGGTCTATAACCTGG GGCATTCCACCATTATGCAGTCAGCGTGGAAACGCGGTCAGAATGTGACCATTCACGGCTGGGCGTACAGTATCAATGATGGCCTGCTGCGCGAT CTTGACGTCACCGCCACCAACAGAGAAACGCTGGAGAACGGCTATCATAAGGGTATTTCCGCCCTAAGTCTGAAATACATTCCCCACCAATAA

Contains a total of 663 characters (nucleotides) which is reflected along the Secondary PCR band in relation to the 100 bp ladder.

Week 6
Worked on ProtocolPCR_PrimerOverlap: consisted of PCR 1' and Second PCR; however, after removal of Gel Electrophoresis from the tray, my gel broke. When I get back into the lab, I will re-run the samples because I saved enough in case things went awry during my work. I will upload PCR picture after I re-run the cloning.

Also, my second run of Virtual Refresher is currently running.

Week 5
A continuation of PyMOL Refresher. In addition, I ran first round of Virtual Screening

PyMOL Refresher Results:






Week 3
//In Progress:// Submit DNA Sequencing (needs weekend to run) //Agarose Gel Information:// The Forward and Reverse Primers used to amplify the Purple Protein coding sequence in the pgbr22 plasmid were the M13 Forward and Reverse primers. This adjustment was made due to the fact that the previous 2 PCR runs, using SP6 and T7, were ineffective.
 * Lane 1: SKIPPED
 * Lane 2: 100 bp ladder
 * Lane 3: Sample A
 * Lane 4: Sample B
 * Lane 5: Sample C
 * Lane 6: Sample D

Week 2
//Agarose Gel Information:// A 1 kb DNA ladder was used instead of a 100 bp ladder. In addition, each sample contains the following with only the Restriction Enzyme and corresponding Enzyme Buffer differing:
 * 1.5 ug plasmid ||
 * 2.5 ul 10X Enzyme Buffer ||
 * X ul DDW to 24ul ||
 * 1 ul ~10 Units of Restriction Enzyme (or ~5 Units of each) ||
 * 25 ul Total Volume ||
 * Lane 1: SKIPPED
 * Lane 2: 1 kb DNA ladder
 * Lane 3: Diluted, uncut plasmid
 * Lane 4: Sample A (RE-EcoRI)
 * Lane 5: Sample B (RE-PvuII)
 * Lane 6: Sample C (RE-EcoRI & PvuII)

//Agarose Gel Information:// The Forward and Reverse Primers used to amplify the Purple Protein coding sequence in the pgbr22 plasmid were the SP6 and T7 Promoters, respectively. The second run of PCR yielded the same results, which could suggest contamination of the SP6 and T7 Promoters (if PCR technique was flawless although that is highly unlikely).
 * Lane 1: SKIPPED
 * Lane 2: 100 bp ladder
 * Lane 3: Sample A
 * Lane 4: Sample B
 * Lane 5: Sample C
 * Lane 6: Sample D

Week 1


//Agarose Gel Information:// The Forward and Reverse Primers used to amplify the Purple Protein coding sequence in the pgbr22 plasmid were the SP6 and T7 Promoters, respectively.
 * Lane 1: SKIPPED
 * Lane 2: 100 bp ladder
 * Lane 3: Sample A
 * Lane 4: Sample B
 * Lane 5: Sample C
 * Lane 6: Sample D
 * Lane 7: SKIPPED
 * Lane 8: 100 bp ladder

Comments: Johnny - add in your other gels and be sure to label everything. - Dr. B