[ http://www.evernote.com/pub/sr320/lab]

012309

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

---

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



PGS_WGSalign.pdf

092708

Vibrio 2-D analysis

Spreadheet of MW and PI of all Vp Proteins

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. - sr320 Sep 18, 2008

091208

- I poured a gel for Sam to run the seafan samples. - sr320 Sep 14, 2008

090508

- ran gel of PGS colonies cloned with TA

positives include 4,6,7,8,9,10,12,17

081208

- 2D gel analysis

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. - sr320 Sep 17, 2008

Below is a comparison of the 061908 and 081208 runs

• 061908 2D gel analysis

This was the first attempt. Some Vertical streaking observed. Assumed this was related to non sterile acrylamide. Some possible differences but hard to tell.