IGEM:MIT/2006/Notebook/2006-8-1

to do (am)

 * 1) START ASAP!!!: repeat failed 7/31 digestions -done
 * 2) order new pchA reverse primer (ask Tom for help?)
 * 3) test osmY+E0840(A,B,C) part with plate reader

to do (pm)

 * 1) nanodrop and run a gel of digest products (NO GEL EXTRACTION)
 * 2) do ligations using old 7/31 gel extracts
 * 3) do ligations using new 8/1 digest products
 * 4) transform everything into new top10 cells

Miniprepped LCs
30-ATF1 ABC and osmY-E0840 ABC and R0040

Run Gel of BAT2 and THI3 PCR Products
Nothing showed (including + control)- so we will PCR BAT2 and THI3 again with 1 &mu;L yeast genomic dna

PCR BAT2 and THI3 (2nd attempt)

 * Used 1&mu;L of Yeast genomic DNA from Samantha...and it worked!! Tomorrow we will PCR-cleanup, cut, and ligate into pSB1A3.  Then we'll be ready to make the isoamyl alcohol generating device! yay!!!!!! :-D

Fax Dr. Schweizer for pUCP18 etc.

 * 1) original fax number is most likely dead or old
 * 2) found 2 new fax numbers and successfully sent a fax to University of Calgary and Dr. Schweizer's updated office fax

Reminder: Cut Pieces We Have

 * 1) B0015: EX
 * 2) B0030: SP
 * 3) B0032: SP
 * 4) R0040:SP (didn't show with 5&mu;L loaded on wide lane gel, but could still be there)
 * 5) More on topical index

Gel extract fragments (7/31)

 * 1) RBS30A-CDS-Term: XP
 * 2) RBS30B-CDS-Term: XP
 * 3) RBS30C-CDS-Term: XP
 * 4) RBS32C-ATF1mut: ES
 * 5) RBS32A-ATF1mut: ES
 * 6) RBS30A-ATF1mut: ES
 * 7) RBS30B-ATF1mut: ES
 * 8) E0840: XP

9 ligations using gel extracts 1-8 (7/31) above

 * 1) R0040+1->Prom-RBS30A-CDS-Term
 * 2) R0040+2->Prom-RBS30B-CDS-Term
 * 3) R0040+3->Prom-RBS30C-CDS-Term
 * 4) 4+Term->RBS32C-ATF1mut-Term
 * 5) 5+Term->RBS32A-ATF1mut-Term
 * 6) 6+Term->RBS30A-ATF1mut-Term
 * 7) 7+Term->RBS30B-ATF1mut-Term
 * 8) R0040+8(E0840)->CP-RBS-GFP-Term
 * 9) osmY+2->osmY-RBS30B-CDS-Term

Take 2 Digestions (same as 7/31)

 * 1) note number system!
 * 2) *<1>: RBS30A-CDS-Term: XP ***this got really messed up because the number 1 looks like the number 7***
 * 3) *<2>: RBS30B-CDS-Term: XP
 * 4) *<3>: RBS32A-CDS-Term: XP
 * 5) *<4>: RBS32C-CDS-Term: XP
 * 6) *<9>: RBS30A-ATF1mut: ES
 * 7) *<10>: RBS30B-ATF1mut: ES
 * 8) *<10***>: RBS30C-ATF1mut: ES
 * 9) *<12>: ATF1mutB-Term: XP
 * 10) *<13>: ATF1mutc-Term: XP


 * Order of lanes: 2, 3, 4, RBS32C-ATF1mut: ES, 9, 10


 * Order of lanes: 10***, 12, 13, 14

13 Take 2 ligations

 * 1) R0040 + <1> ***BAD...the "1" was really "7"; didn't transform this***
 * 2) R0040 + <2>
 * 3) R0040 + <3>
 * 4) R0040 + <4>
 * 5) osmY + <1> ***BAD...the "1" was really "7"; didn't transform this***
 * 6) osmY + <4>
 * 7) <9> + B0015
 * 8) <10> + B0015
 * 9) <10***> + B0015
 * 10) B0030 + <12>
 * 11) B0030 + <13>
 * 12) B0032 + <12>
 * 13) B0032 + <13>

transformations (@ 5:30 pm)

 * 1) prepare 23 plates, review the resistance of backbones
 * 2) *everything in B0015 backbone is Amp/Kan!! rest are just Amp
 * 3) use fresh Top10 cells, pUC19 control, 200 &mu;L