Steven's+Notebook

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103008
Using Go-Taq, PCR'd using aldolase primers, 48 yeast colonies for plate Q1 and 48 colonies from plate Q2. Insight from experience. Do not use toothpicks, foil and PTC-100 (aka broken lid machine) not compatible. about 10% of the of interaction were aldolase. //Will likely develop highthroughput screen using CYTO and 3 sets of primers.//

100808
PGS Analysis Seems to be some sequencing errors on the PGS_wd100708

>PGS_wd100808 translate in frame 0 geneious (1 - NCBI) AGCAGTGGTATCAACGCAGAGTACGCGGGGAGTTCTTCACAGGTTGTATTTCACGTGGGTGTTTCTGTTTTA GATCTTAGGAACGTGGTTAAAAAGTGTGCAATCGGACGGACTGATATATCTATTTGAAGGTGTTTCTCGATC ACTGTACCTTTCATCGGCACATTTAGTAGGTGTGAGGACTCACGTTTTTGAGGATTGTGTCCCCCACTAGGC ACCCAAAGAAATCCGTGAAGCTGACCGGCTGTCCCTGGCGCCTCTTCTGGACAATTCTGGAGACATAAGTGT ACAGACTCTGTGGTTTCAGCAGCCAAAAAGGACTGTGTTCTATATTTCTACCTAGCTCCTCTCGGAGTTCCC CTGGTGTTTTCTGTGGCCAGTCTGGGTGAGAGTAAAGCTGAGACAAATACCTGCCAAATAAAAGTACCGATA CAGTACATTAAAATGTGGTGGAGGGCAGTTTTACTGGTCAGTTTCATAGCTCCTGCACTAGTTTCAGGTGGC CATAACCCTTGTTGTTCATACCCTTGTGAAAACAATGGAGTATGCATGACCCAAGGANGAAATTATTACTGC GACTGTGCAAATCTGGACTTCTATGGCAAACACTGTGAAATTCCTACCTTTATGAAGAAGATCAAGCTGTTT TTAAAACCAAGTCAAAGCACTCTTCATAACCTGCTGACATCCAAAAAATGGCTGTGGAATATCGTCAACAGC GTTCCATTTCTGGCCAATGCAGTGATGAAAAAAGTTTATACCATGAGGTCAGATATGATAGACAGTCCACCA ACCTATACCTCTGAGCACGAATACATCACAGTGGATGCTAATTTCAACTATAGTGTCTACACACGTACCTTG CCTCCAGTACCAAAGGAATGCCCGACACCTATGGGAGTATGGGGAAAGAAGGTGCTACCTCCTGCTGAAGAC ATTGTGAAGGCTTTCTTCATCAGAAAGGAATTCATTCCCGAGCCGTTAGGGACGAGTGCCTTATTTCCTTAC TTTGCTCAGCACTTCACTCACATGTTCTTCAAGACTGACCTAAAGAAAGGGCCGCAGTTTCAGTGGGGAGGA CATGGGGTAGACGTTTCTCATGTATATGGAAAGGACAGAGRTTCYGAGAATAACCTAAGATCTTTCAAAGAT GGGAAACTCAAATTCCAGATCATAGATGGAGAAGAGTGGCCCCCCTTGACTTCTGAAGCAGATGTTCCAATG GGATATCCAAAGGGAATGGAAAAGACCAAGCAATTTGCTCTGGGACACTCCTTCTTTGGTCTACTACCAGGA TTGTTTATGTATGGCACAATCTGGTTACGAGAACATAACAGAGTGTGTGACATCTTGAAAAAGGAGCATCCA GAATGGAATGATGAGAGATTATTTCAAACTGCCAAGCTGGTGATTCTAGGAGAGACCATTAAAATTGTTATA GAGGACTATGTAAAACATCTCAGCAACTATAATTATGATTTGATCTTTAATCCTGAATTGCTGTTTGGCGAG CCATTCCAGTATCAGAACAGGATTGCTTTAGAATTCAACCATCTTTATCACTGGCATCCCCTGATGCCAGAT GAGATTAACATCAGTGGAACTATGTACAATCTAAAAGAGTTCATGTTTCATCCAGAGCTTGTTGTAAAACAT GGAATGAGGAACTTTGTGGATTCTCTGTCCAAACAGAGAGCTGGATTATTTGGACCCCGAAATCATGGCCCT GTGACAATTCCTGTGGTGACAGAATTGATCAAACATGGCCGCACCTTAAGGCTGCAGTCATTCAACCAGTAT AGAAAGAGATTTGACATGGAGCCTATCAAGTCTTTTGAAGAACTGACAGGAGAGAAGAAAATGGCCAAGCAA CTTGAGGATTTCTATGGTGATGTTAATGCTGTGGAGTTTTATGTAGGATTGATAATGGAGAAAAGAAGACAC AATTCTATGTTTGGTGACTCTTTGGTACAAATTGGTGCCCCGTATTCTGTGAAGGGTCTGATGGCAAATCCT ATYTGTAGTCCCAAGTACTGGAAAYCTTCTACGTTTGGTGGAGAGGTTGGGTTCAACATAGTAAAAACTTCT TCACTTAAAAANCTGTTTTGTGAAAATATCAAAGGAGAATGTCCATTAGTATCATTTAGAGTCCCAGACTAT GTTGAGGGAGATGTWACAGAATTCATCAACCAAAAACTAGAATTATAACACTRCATTTCATTTYAATGGCAT TGTGTTTAAACAAAACYTTMGTAAAGCATTTCAAATGTTGTCATGAAATTTGACTGATATTGTACTGTGAGT GTGTAAATGCTTAGTTATGCATTATTTTCATTTTTAAAAATCACTTTCTGAATTTAGTAGCATTTAATGTGA ATAATGTCTATTAATATTTTTATAGATGTTTTGAATTTGAAATCTGAAATCCAGTATCATTTTA

100708
PGS Analysis

[[image:Picture_5.png width="817" height="157"]]
Resulting Sequence >PGS_wd100708 AAGCAGTGGTATCAACGCAGAGTACGCGGGGAGTTCTTCACAGGTTGTATTTCACGTGGGTGTTTCTGTTTT AGATCTTAGGAACGTGGTTAAAAAGTGTGCAATCGGACGGACTGATATATCTATTTGAAGGTGTTTCTCGAT CACTGTACCTTTCATCGGCACATTTAGTAGGTGTGAGGACTCACGTTTTTGAGGATTGTGTCCCCCACTAGG CACCCAAAGAAATCCGTGAAGCTGACCGGCTGTCCCTGGCGCCTCTTCTGGACAATTCTGGAGACATAAGTG TACAGACTCTGTGGTTTCAGCAGCCAAAAAGGACTGTGTTCTATATTTCTACCTAGCTCCTCTCGGAGTTCC CCTGGTGTTTTCTGTGGCCAGTCTGGGTGAGAGTAAAGCTGAGACAAATACCTGCCAAATAAAAGTACCGAT ACAGTACATTAAAATGTGGTGGAGGGCAGTTTTACTGGTCAGTTTCATAGCTCCTGCACTAGTTTCAGGTGG CCATAACCCTTGTTGTTCATACCCTTGTGAAAACAATGGAGTATGCATGACCCAAGGANGAAAATTATTACT GCGACTGTGCAAATCTGGACTTCTATGGCAAACACTGTGAAATTCCTACCTTTATGAAGAAGATCAAGCTGT TTTTAAAACCAAGTCAAAGCACTCTTCATAACCTGCTGACATCCAAAAAATGGCTGTGGAATATCGTCAACA GCGTTCCATTTCTGGCCAATGCAGTGATGAAAAAAGTTTATACCATGAGGTCAGATATGATAGACAGTCCAC CAACCTATACCTCTGAGCACGAATACATCACAGTGGATGCTAATTTCAACTATAGTGTCTACACACGTACCT TGCCTCCAGTACCAAAGGAATGCCCGACACCTATGGGAGTATGGGGAAAGAAGGTGCTACCTCCTGCTGAAG ACATTGTGAAGGCTTTCTTCATCAGAAAGGAATTCATTCCCGAGCCGTTAGGGACGAGTGCCTTATTTCCTT ACTTTGCTCAGCACTTCACTCACATGTTCTTCAAGACTGACCTAAAGAAAGGGCCGCAGTTTCAGTGGGGAG GACATGGGGTAGACGTTTCTCATGTATATGGAAAGGACAGAGRTTCYGAGAATAACCTAAGATCTTTCAAAG ATGGGAAACTCAAATTCCAGATCATAGATGGAGAAGAGTGGCCCCCCTTGACTTCTGAAGCAGATGTTCCAA TGGGATATCCAAAGGGAATGGAAAAGACCAAGCAATTTGCTCTGGGACACTCCTTCTTTGGTCTACTACCAG GATTGTTTATGTATGGCACAATCTGGTTACGAGAACATAACAGAGTGTGTGACATCTTGAAAAAGGAGCATC CAGAATGGAATGATGAGAGATTATTTCAAACTGCCAAGCTGGTGATTCTAGGAGAGACCATTAAAATTGTTA TAGAGGACTATGTAAAACATCTCAGCAACTATAATTATGATTTGATCTTTAATCCTGAATTGCTGTTTGGCG AGCCATTCCAGTATCAGAACAGGATTGCTTTAGAATTCAACCATCTTTATCACTGGCATCCCCTGATGCCAG ATGAGATTAACATCAGTGGAACTATGTACAATCTAAAAGAGTTCATGTTTCATCCAGAGCTTGTTGTAAAAC ATGGAATGAGGAACTTTGTGGATTCTCTGTCCAAACAGAGAGCTGGATTATTTGGACCCCGAAATCATGGCC CTGTGACAATTCCTGTGGTGACAGAATTGATCAAACATGGCCGCACCTTAAGGCTGCAGTCATTCAACCAGT ATAGAAAGAGATTTGACATGGAGCCTATCAAGTCTTTTGAAGAACTGACAGGAGAGAAGAAAATGGCCAAGC AACTTGAGGATTTCTATGGTGATGTTAATGCTGTGGAGTTTTATGTAGGATTGATAATGGAGAAAAGAAGAC ACAATTCTATGTTTGGTGACTCTTTGGTACAAATTGGTGCCCCGTATTCTGTGAAGGGTCTGATGGCAAATC CTATYTGTAGTCCCAAGTACTGGAAAYCTTCTACGTTTGGTGGAGAGGTTGGGTTCAACATAGTAAAAACTT CTTCACTTAAAAAANCTGTTTTGTGAAAATATCAAAGGAGAATGTCCATTAGTATCATTTAGAGTCCCAGAC TATGTTGAGGGAGATGTWACAGAATTCATCAACCAAAAACTAGAATTATAACACTRCATTTCATTTYAATGG CATTGTGTTTNAAACAAAACYTTMGTAAAGCATTTCAAATGTTGTCATGAAATTTGACTGATATTGTACTGT GAGTGTGTAAATGCTTAGTTATGCATTATTTTCATTTTTAAAAATCACTTTCTGAATTTAGTAGCATTTAAT GTGAATAATGTCTATTAATATTTTTATAGATGTTTTGAATTTGAAATCTGAAATCCAGTATCATTTTA



092708
Vibrio 2-D analysis

[|Spreadheet of MW and PI of all Vp Proteins] media type="googlespreadsheet" key="pyc8PVwzKMBpxNq00ZCmVAw" width="500" height="300"

092608
SeaFan Seq analysis

Gel from Sam

//Sent off// New-SF-1-9.11.08_LABY_A.ab1 391 New-SF-1-9.11.08_LABY_Y.ab1 0 New-SF-13-9.12.08_LABY_A.ab1 393 New-SF-13-9.12.08_LABY_Y.ab1 723 New-Spot-9-9.11.08_LABY_A.ab1 393 New-Spot-9-9.11.08_LABY_Y.ab1 790 Old-SF-1-9.12.08_LABY_A.ab1 48 Old-SF-1-9.12.08_LABY_Y.ab1 802 SF-4-9.10_LABY_A.ab1 30 SF-4-9.10_LABY_Y.ab1 389 SF-6-9.10_LABY_A.ab1 390 SF-6-9.10_LABY_Y.ab1 661 SF-8-9.10_LABY_A.ab1 0 SF-8-9.10_LABY_Y.ab1 297

//Nice stuff back from// SF #1 (New-SF-1-9.11.08_LABY_A) SF #13 (New-SF-13-9.12.08_LABY_A; New-SF-13-9.12.08_LABY_Y.) Spot #9 (New-Spot-9-9.11.08_LABY_A; New-Spot-9-9.11.08_LABY_Y) SF #1 Original Set (Old-SF-1-9.12.08_LABY_Y) SF #4 (SF-4-9.10_LABY_Y) SF #6 (SF-6-9.10_LABY_Y) SF #8 (SF-8-9.10_LABY_Y)



old data

>006_C01_SEAFAN1.ab1 289 CCACTATTTAGTAGGCTGAGGTCTCGTTCGTCAATGGAATTAACCAGACA AATCACTCCACCAACTAAGAACGGCCATGCACCACCACCCATAGAATCAA GAAAGAGCTCTCAATCTGTCAATCCTTACTATGTCTGGACCTGGTAAGTT TTCCCGTGTTGAGTCAAATTAAGCCGCAGGCTCCACTCTTGGTGGTGCCC TTCCGGCAATTCCTATAACTATCTACCCTGCTACAATACTCC >007_B01_SEAFAN5.ab1 263 GAGGTCTCGTTCGTCAACGGAATTAACCCGACTAATCCCTCCACCTACTA ACAGCGGTCCTGCACCACCACCCATATCATCAAGAAAGAGCTCTCCTTCT GTCCCTCCTTATTATGGCTGGACCTGGTAAGTTTTCCCGTGTTGAGTCTA ATTAAGCCGCAGGCTCCCCTCCTGGTGGTGTCCTTCCGTCACTTCCTTTA AGTTTCCTCCTTGCGACCATACTCCCCCCACAACCCAAATACTTTGATTT CTCATGAGGTGCT >008_A01_SEAFAN7.ab1 218 TATTTAGCAGGCTGAGGTCTCGTTCGTCAACGGAATTAACCAGACAAATC ACTCCACCAACTAAGAACGGCCATGCACCACCACCCATAGAATCAAGAAA GAGCTCTCAATCTGTCAATCCTTACTATGTCTGGACCTGGTAAGTTTTCC CGTGTTGAGTCAAATTAAGCCGCAGGCTCCACTCCTGGTGGTGCCCTTCC GTCAATTCCTTTAAGTTT >009_H03_SEAFAN8.ab1 221 TTTCGAATATGCACACTATTTAGCAGGCTGAGGTCTCGTTCGTCAATGGA ATTAACCAGACAAATCACTCCACCAACTAAGAACGGCCATGCACCACCAC CCATAGAATCAAGAAAGAGCTCTCAATCTGTCAATCCTTACTATGTCTGG ACCTGGTAAGTTTTCCCGTGTTGAGTCAAATTAAGCCGCAGGCTCCACTC CTGGTGGTGCCCTTCCGTCAA



091908
Run gel from 091808. New Vts and Dungans. Vt P, Flim, H, M, mix primers

cut ~250 bp Flim

Dungans A - XCVC 19t 8/7 B - XCVC 12T C - XCVC 17t 8/11 D - VNTC 12c1 G10 7/25 E - XCVE 13T F - VATM 1.2 T 7/14 G - BC05CA 18T H5 7/18 H - VNTC 1.5T 7/14

cut bands for all except "B"

091808
- PCR Dungans, 50ul [|Immunomix], 4ul template, 56C. Did loading in hood to decrease possible carryover.

- redo new Vt primers pooling cDNA t=24 auto oyster, t=24 sterile water, t-2hr SW ph 7.57, t=2hr SW ph 8.57 50ul [|Immunomix], 3ul template, 50C.

091708
- Ran new Dungans with a re-heat, 50ul [|Immunomix], 4ul template, 50C.

Ran 3 new Vt primers + FliM. Pooled ~8 1:4 cDNA dilutions from pH and exposures. 50ul [|Immunomix], 3ul template, 50C. Analysis: Vt : Fllim had ~3 bands. ~400, ~800, ~1500 Will red with different template & do a primer mix.

Dungans all had products, however not happy with bands in negative. redo. 1221762102

091208
- I poured a gel for Sam to run the seafan samples. 1221425834

090508
- ran gel of PGS colonies cloned with TA positives include 4,6,7,8,9,10,12,17

081208
- 2D gel analysis media type="custom" key="1996962" 2nd run (same sample as 061908). Horizontal streaking. This time additional samples were run during IEF. Assumed might need to IEF longer. On 091608, third run with same sample. Other issues surround that run. 1221663662

Below is a comparison of the 061908 and 081208 runs media type="custom" key="1998766"

media type="custom" key="1996970" This was the first attempt. Some Vertical streaking observed. Assumed this was related to non sterile acrylamide. Some possible differences but hard to tell.
 * 061908 2D gel analysis