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 μL yeast genomic dna

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 5μL loaded on wide lane gel, but could still be there)
5. More on [[../../Index | topical index]]

Gel extract fragments (7/31)

1. RBS_30A-CDS-Term: XP
2. RBS_30B-CDS-Term: XP
3. RBS_30C-CDS-Term: XP
4. RBS_32C-ATF1mut: ES
5. RBS_32A-ATF1mut: ES
6. RBS_30A-ATF1mut: ES
7. RBS_30B-ATF1mut: ES
8. E0840: XP

ligations using gel extracts 1-8 above

1. R0040+1->Prom-RBS_30A-CDS-Term
2. R0040+2->Prom-RBS_30B-CDS-Term
3. R0040+3->Prom-RBS_30C-CDS-Term
4. 4+Term->RBS_32C-ATF1_mut-Term
5. 5+Term->RBS_32A-ATF1_mut-Term
6. 6+Term->RBS_30A-ATF1_mut-Term
7. 7+Term->RBS_30B-ATF1_mut-Term
8. R0040+8(E0840)->CP-RBS-GFP-Term
9. osmY+2->osmY-RBS_30B-CDS-Term

Take 2 Digestions (same as 7/31)

1. 30B 32A 32C: RCT
2. 32C 30A 30B: 30C RA
3. B C: AT
4. R0040

transformations (@ 2:30 pm)

1. prepare 9 plates, review the resistance of backbones
2. use fresh Top10 cells, pUC19 control, 200 μL