Julia+C.

Friday 12/6 Ran Inhibition assay. During the first trial, forgot to add inhibitor and positive orthovanidate control. Second trial, added inhibitor, but still forgot the orthovanidate control. Trial 1:

Trial 2:

Also attempted to do virtual screening with the control ligands. Did not run. Wednesday 11/27 Concentrated both elutions to 4.26 mg/ml in concentration tube and ran through FPLC. Figure above: FPLC result for YopH

Tuesday 11/26 Purified Trial 2, nanodropped, and ran on PAGE gel Color Plus prestained Protein Ladder.

Figure above: SDS-PAGE gel of large scale expression of YopH, flask 1 Figure above: SDS-PAGE gel of large scale expression of YopH, flask 1

Monday 11/25 Sonicated the two samples, spun down and saved supernatant. Purified Trial 1 using a used chromatography tube.

Friday 11/22 The next day, transferred to large tubes and centrifuged to retreive pellets, which were resuspended in sonication buffer and put into conical tubes.

Thursday 11/21 Started Option A expression from an available YapH plate to be used as a surrogate. Grew up 2 separate tubes of colonies in the shaking incubator for 8 hours, then measured the OD600 using a spectrophotometer. It never got up to 0.1. Left overnight for 18 hours in biobricks lab in room temperature incubator. I have now realized that the main reason the OD600 never reached the proper values is because instead of growing one of the tube colonies in large scale growth and adding in aliquots of the second tube to reach the proper OD600, we grew up two huge cultures in 2-2L flasks.

Wednesday 11/20 Mini-prepped last batch.

Tuesday 11/19 Made 1 more batch of tubes. Negative results for second set of colonies. The colonies in the tubes grew very fitfully and therefore had very low concentrations when midiprepped.

Friday 11/15 Miniprepped second batch of tubes and received negative results from the first batch.

Thursday 11/14 Remade batch of tubes with Kanomycin. Made a list of negative and positive control ligands for virtual.

Wednesday 11/13 Mini-prepped samples and submitted to sequencing.

Sunday 11/10 Decanted and centrifuged tubes, and then realized that I had FORGOTTEN TO ADD KANOMYCIN.

Saturday 11/9 Plate A and Plate B grew small yellow colonies, but B had more colonies. Made 8 tubes and a master plate from the two plates (#1-4 from plate A and #5-8 from plate B)

Friday 11/8 Cohesive end generation for PCR inserts and Accepting vector. Annealing and Transformation Tube A - 2 ul accepting, 4 ul insert Tube B- 2 ul accepting, 6 ul insert

Wednesday 11/6 PCR cleanup - 43.0 ng/ul

Tuesday 11/5 Prepped pNIC with BSAI-HF, made 2-40 microliter tubes of prepped plasmid.

Friday 11/1/13 Midi prepped pNIC, but achieved low concentration of 7.9 and 8 ng/microliter, so moved on to using Alyssa's prepped plasmid.

Friday 10/25 Centrifuged and decanted pNIC - made 4 tubes.

Thursday 10/24 Virtual setup. Not yet complete. Restarted pNIC protocols- overnight culture with 160 mL of LB.

Friday 10/18 PCR cleanup on both plasmid and insert. Messed up plasmid PCR cleanup by decanting during the last step, but PCR squared came out fine with a concentration of 58.7 ng/microliter. Nanodropped both. Figure above: Nanodrop of remaining PCR-cleaned up pNIC plasmid Figure above: Nanodrop of PCR-cleaned up PCR squared

Thursday 10/17 Prepared pNIC accepting vector with restriction enzymes

Tuesday 10/15 Midi prepped pNIC Average concentration: 39.7 g/Microliter Figure above: Lab notebook picture of Midiprep results

Friday 10/11 Took out and centrifuged pNIC Figure 1 above: Overnight incubation of pNIC Figure 2 above: Centrifuged and decanted pNIC

Thursday 10/10 Tested PCR squared with Fermentas gene ruler, SUCCESS!! Put pNIC transformation in incubator overnight Figure above: 0.7 agarose gel with 100 bp Fermentas gene ruler and PCR squared samples.

Great work updating your wikispace Julia. The captions and analysis look good, but make sure you upload the images properly I cant seem to find them. Where are you exactly with virtual? Thank you. -Max 10/21/13 Tuesday 10/8 Made PCRsquared at 60 degrees and middle times.

Monday 10/7 Made and ran gel of SPCR at 60 degrees and middle times. Success! But PCR tube was crushed by the lid being too tight. Figure above: Secondary PCR attempt at 60 degrees and middle times. First band: 100 bp ladder. Second band: Visible SPCR smear.

Wednesday 10/2 Ran gel of SPCR, which failed to show bands. Figure above: Secondary PCR attempt at 56 degrees. First band: 1kbp ladder.

Tuesday 10/1 Ran PPCR gel- band was formed. Made and left SPCR (56 degrees) Figure above: Primary PCR attempt at 56 degrees: 1st band: 100 bp ladder. 2nd band: successful sample smear.

Monday 9/30 Remade PPCR mix at 56 degrees and ran overnight.

Saturday 9/28 Made SPCR and ran on gel, which tore but also didn't show any bands. Finished Pymol Refresher Figure above: Secondary PCR attempt. 1st band: 100 bp ladder.

Friday 9/27 Ran PPCr again, smear was present Figure above: Primary PCR attempt. 1st band: 100 bp ladder. 3rd band: PPCR. Thursday 9/26 Made LB Media and attempted Primary PCR, which didn't show any bands or even the ladder.

Julia, you're missing week 5 and 6 :( update your wikispaces! - Michael T.

Week 3 & 4 Julia - ok, need Primary PCR (for your next Wiki), Crop your gel images in the Imaging Software, Add captions to bottom of Figures, Neeed brief analysis, include an image of ladder. - Dr. B 092713

Monday 9/16/13 Completed enzyme digest protocol, and didn't achieve desired results. Lanes 4 and 5 were very faint and did not have the correct amount of lines, meaning the digest did not go to completion. Figure 1: pGBR22 Enzyme digest gel run. Lane 1: empty. Lane 2: 1kb DNA ladder. Lane 3: EcoRI digest. Lane 4: PvuII digest. Lane 5: EcoRi and PvuII digest. Lane 6: Undigested pGBR22. Designed Tail primers for pNIC-Bsa4 Cloning Final result: TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTT TGTTTAACTTTAAGAAGGAGATATACATATGCACCATCATCATCATCATTCTTCTGGTGT AGATCTGGGTACCGAGAACCTGTACTTCCAATCCATGGACGCGCTGACCACCCTCCCGATCAAAAAGCACACCGCGC TGCTGAACCGTTTCCCG GAAACCCGCTTCGTTACCCAACTGGCGAAAAAGCGTGCGTCTTGGATCGTTTTCGGTCAC TACCTCACTCCAGCACAGTTTGAAGATATGGATTTTTTCACCAATCGTTTCAATGCGATC CTGGACATGTGGAAAGTTGGCCGTTACGAAGTTGCGCTGATGGACGGTGAACTGACCTCT GAACACGAAACCATCCTGAAAGCGCTGGAACTCGACTACGCTCGCATCCAGGACGTTCCA GACCTCACCAAACCGGGCCTGATCGTTCTCGACATGGACTCTACCGCTATCCAGATCGAA TGCATCGACGAAATTGCGAAGCTGGCGGGTGTTGGCGAGGAAGTGGCCGAAGTTACGGAA CGTGCGATGCAGGGCGAGCTGGACTTCGAACAGTCTCTGCGTCTGCGTGTTTCTAAACTC AAAGACGCCCCTGAACAGATCCTGAGCCAGGTTCGTGAAACGCTGCCGCTCATGCCTGAA CTGCCGGAACTGGTTGCGACCCTGCACGCGTTCGGTTGGAAGGTAGCAATCGCGTCTGGT GGTTTCACCTACTTTTCTGACTACCTGAAGGAACAACTCAGCCTCGATTACGCGCAGTCT AACACCCTGGAAATTGTTTCTGGTAAACTGACTGGTCAAGTTCTGGGTGAAGTTGTGTCT GCTCAGACCAAAGCGGACATCCTGCTGACCCTGGCGCAACAGTACGACGTTGAAATCCAC AACACCGTTGCGGTGGGTGACGGTGCGAACGACCTGGTTATGATGGCGGCTGCGGGCCTC GGTGTAGCGTACCATGCGAAACCGAAGGTTGAGGCGAAGGCGCAGACCGCAGTTCGTTTC GCTGGTCTCGGTGGTGTCGTTTGCATCCTGTCTGCGGCGCTCGTTGCGCAGCAAAAACTC TCTTGGAAATCTAAACCGTAACAGTAAAGGTGGATACGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCA CTCGAGCACCACCACCACCACC ACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTG AGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGA AAGGAGGAACTATATCCGGATTGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGC GGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGC TCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCT AAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAA ACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCC TTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACT CAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTG GTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTT TACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTT CTAAATACATTCAAATATGTATCCGCTCATGAATTAATTCTTAGAAAAACTCATCGAGCA TCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCC GTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGT ATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAA AAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCA AAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAA AATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATA CGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACA CTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATG CTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAAT GCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTG TAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCT TCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTAT ACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCC GTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTG TTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAA AAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACA AAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTT CCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCG TAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATC CTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGA CGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCC AGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGC GCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACA GGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGG TTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTA TGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCT CACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAG TGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAA GCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGC ATATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACAC TCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGA CGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTC CGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCG GTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTC CAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTT AAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCAT GGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGA ACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGA CCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCC ACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGA CTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCA GGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATT CTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGAT CATGCGCACCCGTGGGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTT GGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAG CGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAG CGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGAC GATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCAT CGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACT GCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGC GGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGG GCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGC TGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATG AGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGG ACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAG TGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCC AGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGC CAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCT GGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAA TAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGC AGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCAC TGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTT CTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGA CAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACT GTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCG CTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAA CGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACAT TCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGC GCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAG CAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAG GAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAA GCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAG GCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGG ATCGAGATCTCGATCCCGCGAAAT

9/20/13 Prepared Oligo mix of V. cholarae DNA. Will start primary and secondary PCR next week.

Week 1 & 2 Julia, ok looks good. paste aa sequence as text only (no code). Say what is in each lane for this PCR. Dr. B 090913

8/30/13 Today I was assigned my target: phosphoserine phosphatase (Vibrio cholerae) Gene: YP_005334106.1

The aa sequence was found on NCBI, so it was different than the original information page on wikispaces. code format="genbank" 1 mdalttlpik khtallnrfp etrfvtqlak kraswivfgh yltpaqfedm dfftnrfnai 61 ldmwkvgrye valmdgelts ehetilkale ldyariqdvp dltkpglivl dmdstaiqie 121 cideiaklag vgeevaevte ramqgeldfe qslrlrvskl kdapeqilsq vretlplmpe 181 lpelvatlha fgwkvaiasg gftyfsdylk eqlsldyaqs ntleivsgkl tgqvlgevvs 241 aqtkadillt laqqydveih ntvavgdgan dlvmmaaagl gvayhakpkv eakaqtavrf 301 aglggvvcil saalvaqqkl swkskp code I ordered the oligos under the order VcPP_1. The modified sequence was:

1 ATGGACGCGCTGACCAC 17 2 CCGGGAAACGGTTCAGGAGCGCGGTGTGTTTTTTGATCGGCAGCGTGGTCAGCGCGTCCA 60 3 TCCTGAACCGTTTCCCGGAAACCCGTTTCGTTACGCAACTGGCGAAGAAACGCGCGAGCT 60 4 TCTTCAAACTGTGCCGGGGTCAGATAGTGACCGAAAACGATCCAGCTCGCGCGTTTCTTC 60 5 CCCGGCACAGTTTGAAGATATGGACTTCTTCACCAATCGCTTTAATGCCATCCTCGATAT 60 6 CGTCCATGAGCGCAACCTCATAACGACCCACTTTCCACATATCGAGGATGGCATTAAAGC 60 7 GGTTGCGCTCATGGACGGTGAACTCACCTCTGAACACGAAACCATTCTGAAGGCGCTGGA 60 8 TTGGTGAGGTCCGGAACGTCCTGGATACGTGCGTAATCGAGTTCCAGCGCCTTCAGAATG 60 9 CGTTCCGGACCTCACCAAACCGGGTCTCATCGTTCTGGACATGGATTCTACCGCGATTCA 60 10 ACACCCGCCAGCTTCGCGATTTCGTCGATGCATTCGATCTGAATCGCGGTAGAATCCATG 60 11 CGAAGCTGGCGGGTGTCGGTGAGGAAGTTGCGGAAGTTACCGAACGTGCTATGCAGGGCG 60 12 CAGTTTAGAAACACGGAGACGCAGAGACTGTTCGAAATCCAGTTCGCCCTGCATAGCACG 60 13 CGTCTCCGTGTTTCTAAACTGAAGGATGCACCGGAACAGATCCTGAGCCAAGTTCGTGAA 60 14 GTCGCAACGAGCTCTGGCAGTTCCGGCATCAGCGGCAGGGTTTCACGAACTTGGCTCAGG 60 15 CCAGAGCTCGTTGCGACCCTGCACGCATTCGGTTGGAAGGTAGCAATCGCCTCCGGTGGT 60 16 CCAGAGAGAGCTGCTCTTTCAGGTAGTCGCTGAAGTAGGTAAAACCACCGGAGGCGATTG 60 17 GAAAGAGCAGCTCTCTCTGGACTATGCGCAGTCTAACACCCTCGAAATTGTTTCTGGTAA 60 18 GCGCGGAGACAACTTCACCGAGAACCTGACCAGTGAGTTTACCAGAAACAATTTCGAGGG 60 19 GTGAAGTTGTCTCCGCGCAGACCAAAGCGGACATCCTGCTGACGCTCGCCCAGCAGTATG 60 20 TTCGCGCCATCGCCAACCGCAACGGTGTTGTGAATTTCAACGTCATACTGCTGGGCGAGC 60 21 TGGCGATGGCGCGAACGACCTGGTTATGATGGCGGCTGCGGGCCTGGGTGTGGCCTACCA 60 22 ACGAACCGCCGTTTGCGCCTTTGCTTCGACTTTCGGTTTCGCGTGGTAGGCCACACCCAG 60 23 GCAAACGGCGGTTCGTTTTGCGGGTCTGGGTGGTGTCGTTTGTATTCTGTCTGCCGCGCT 60 24 TTACGGTTTAGATTTCCAAGACAGTTTTTGCTGCGCCACCAGCGCGGCAGACAGAAT 57

Week 2 9/02/13 Today I worked on 3 labs: Quantifying DNA using Nanodrop, Submitting DNA to DNA Sequencing Facility, and I started the PCR Protocol

__**Results for Nanodrop:**__

__**The results from the sequencing facility:**__ Forward NNNNNNNNNNNNNNNGCGAANNNNGGCCCGACGTNCGCATGCTCCCGGCCGCCATGGCCGCGGGATTTTAGTTGATGGTGATGGTGATGACCGAGCAAAGAGTGGCGTGCAATGGATATTTCACACTGCTCAACAAATGTGTAATCCTTGTTGTGACTGGTTACATCCAGTTTGCGGTCAACATAGTGATACCCTGGCATCCTCACAGGCTTCTTTGCCTTGTAAGTAGATTTGAATTCACACAAATAGTAACCACCTCCTTCCAACTTCAGAGCCATAAAGTTGTTTCCTATCAGCATTCCATCTCGTGCAAAGAGACGCTCAGTGTTGGGTTCCCAGCCCTGTGTCTTCTTCTGCATAACAGGTCCATTGGGAGGAAAGTTCACACCAGAGATTTTGACATTGTAGATGAAACAGTTGCCTTGGATGCTGGAATCATTGCTGACAGTACACACTGCACCATCTTCAAAGTTCATGATCCTCTCCCATGTATATCCCTCAGGGAATGACTGCTTTACATAATCAGGGATGTCTTCAGGGTACTTGGTGAATGGTATGCTTCCGTATTGAGACAGTGGTGATAAAATATCCCAAGCAAATGGCANANGTCCNNNNNTGGTGACAGTGAGCTTTACCGTCTGCTCCCCCTCGTAAGGCTTTCCTTTTCCATCGCCTTCGACCTCAAAGTANTGTCCATTGACCGTGCCTGACATATAAACCTTGTANGTCATTTGTTTAGCGATCACACTCATGATATTTCTCCTTCAATCAATCAAAATCACTANTGCGGCCGCCTGCAGTCNANCATATGGGAGANCTCCCAACGCGTTGGANGCATAGCTTGANTANTCTATAGTGTCACTAAATAGCTTGCNTATCATGGTCATAGCTGNTTCCTGNGTNAAATTGTATCCNCTCACNATTCNCACACATACGAGCCGNANCNTNANTGNAAAGCTGGGNNGCTATNNNNNNNNACTCACATTANNCNTNNNNNNNCTGCCNCTTCAGTNGGAANCTNTNNGCNNCTGCNTATGANNNCANNNNNGGGANNNNGTTGNNTATGGNNCTNNCNNTCNCNNTNNNTNANTNNNNNNNNNGNNNTCGNTNNNNNNNGNNNNNNNNNNNNNNNNNANNNNNNNNCNNANCNNNNANNNNAAANNNNNNNNANNNNANNNNCNNANNNCNTNNNNNTTCNNNGNNNNCCCNNNNNN Reverse NNNNNNGGGNNNNCNNNGAANNNNNANGNNNTNNGNNNNTNNNNTNNNNNNNNTTTNNNNTNNNNGNTNNGNNNNNNNNTNNNNNNNNNNNNNNNNNCNNNNNNNANCGANNNCNNNNNNNNNANTNANNNANNGNGANNGNNAGNNCCATANNCAACNNNNTCCCNNNNNTGNNNTCATANGCAGNNGCNNANAGNTTCCNACTGAAGNGGCAGNNNNNNNANGNNTAATGTGAGTNNNNNNNNATAGCNNCCCAGCTTTNCANTNANGNTNCGGCTCGTATGTGTGNGAATNGTGAGNGGATACAATTTNACNCAGGAANCAGCTATGACCATGATANGCAAGCTATTTAGTGACACTATAGANTANTCAAGCTATGCNTCCAACGCGTTGGGAGNTCTCCCATATGNTNGACTGCAGGCGGCCGCANTAGTGATTTTGATTGATTGAAGGAGAAATATCATGAGTGTGATCGCTAAACAAATGACNTACAAGGTTTATATGTCAGGCACGGTCAATGGACANTACTTTGAGGTCGAAGGCGATGGAAAAGGAAAGCCTTACGAGGGGGAGCAGACGGTAAAGCTCACTGTCACCANNNNNGGACNTNTGCCATTTGCTTGGGATATTTTATCACCACTGTCTCAATACGGAAGCATACCATTCACCAAGTACCCTGAAGACATCCCTGATTATGTAAAGCAGTCATTCCCTGAGGGATATACATGGGAGAGGATCATGAACTTTGAAGATGGTGCAGTGTGTACTGTCAGCAATGATTCCAGCATCCAAGGCAACTGTTTCATCTACAATGTCAAAATCTCTGGTGTGAACTTTCCTCCCAATGGACCTGTTATGCAGAAGAAGACACAGGGCTGGGAACCCAACACTGAGCGTCTCTTTGCACGAGATGGAATGCTGATAGGAAACAACTTTATGGCTCTGAAGTTGGAAGGAGGTGGTTACTATTTGTGTGAATTCAAATCTACTTACAAGGCAAAGAAGCCTGTGAGGATGCCAGGGTATCACTATGTTGACCGCAAACTGGATGTAACCAGTCACAACAAGGATTACACATTTGTTGAGCAGTGTGAAATATCCATTGCACGCCACTCTTTGCTCGGTCATCACCATCACCATCAACTAAAATCCCGCGGCCATGGCGGCCGGGAGCATGCGNACGTCGGGCCNNNNTTCGCNNNNNNNNNNNNNNN Sequencing results BLASTED against Human:

Sequencing results BLASTED against All:

Results for PCR Protocol:

The pGBR22 gene I sent to sequencing came back with 1,214 base pairs, many of which were labeled N. Taking into account that the PCR process often strips the ends of the DNA, my visible bands were within the ballpark of the expected number of base pairs. (PCR doesn't strip them but rather can start inboard of the start for DNA Sequencing - Dr. B)