IGEM:MIT/2006/Notebook/2006-8-1: Difference between revisions
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==Take 2 Digestions (same as 7/31)== | ==Take 2 Digestions (same as 7/31)== | ||
#<1>: RBS<sub>30A</sub>-CDS-Term: XP | #note number system! | ||
<2>: RBS<sub>30B</sub>-CDS-Term: XP | #*<1>: RBS<sub>30A</sub>-CDS-Term: XP | ||
#RBS<sub>32A</sub>-CDS-Term: XP | #*<2>: RBS<sub>30B</sub>-CDS-Term: XP | ||
#RBS<sub>32C</sub>-CDS-Term: XP | #*<3>: RBS<sub>32A</sub>-CDS-Term: XP | ||
# | #*<4>: RBS<sub>32C</sub>-CDS-Term: XP | ||
#*<9>: RBS<sub>30A</sub>-ATF1mut: ES | |||
#*<10>: RBS<sub>30B</sub>-ATF1mut: ES | |||
#*<10*>: RBS<sub>30C</sub>-ATF1mut: ES | |||
#*<12>: ATF1mut<sub>B</sub>-Term: XP | |||
#RBS<sub>30B</sub>-ATF1mut: ES | #*<13>: ATF1mut<sub>c</sub>-Term: XP | ||
#*10*RBS<sub>30C</sub>-ATF1mut: ES | |||
# | |||
==Take 2 ligations== | ==Take 2 ligations== | ||
Revision as of 12:13, 1 August 2006
to do (am)
- START ASAP!!!: repeat failed 7/31 digestions -done
- order new pchA reverse primer (ask Tom for help?)
- test osmY+E0840(A,B,C) part with plate reader
to do (pm)
- nanodrop and run a gel of digest products (NO GEL EXTRACTION)
- do ligations using old 7/31 gel extracts
- do ligations using new 8/1 digest products
- 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.
- original fax number is most likely dead or old
- 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
- B0015: EX
- B0030: SP
- B0032: SP
- R0040:SP (didn't show with 5μL loaded on wide lane gel, but could still be there)
- More on [[../../Index | topical index]]
Gel extract fragments (7/31)
- RBS30A-CDS-Term: XP
- RBS30B-CDS-Term: XP
- RBS30C-CDS-Term: XP
- RBS32C-ATF1mut: ES
- RBS32A-ATF1mut: ES
- RBS30A-ATF1mut: ES
- RBS30B-ATF1mut: ES
- E0840: XP
9 ligations using gel extracts 1-8 (7/31) above
- R0040+1->Prom-RBS30A-CDS-Term
- R0040+2->Prom-RBS30B-CDS-Term
- R0040+3->Prom-RBS30C-CDS-Term
- 4+Term->RBS32C-ATF1mut-Term
- 5+Term->RBS32A-ATF1mut-Term
- 6+Term->RBS30A-ATF1mut-Term
- 7+Term->RBS30B-ATF1mut-Term
- R0040+8(E0840)->CP-RBS-GFP-Term
- osmY+2->osmY-RBS30B-CDS-Term
Take 2 Digestions (same as 7/31)
- note number system!
- <1>: RBS30A-CDS-Term: XP
- <2>: RBS30B-CDS-Term: XP
- <3>: RBS32A-CDS-Term: XP
- <4>: RBS32C-CDS-Term: XP
- <9>: RBS30A-ATF1mut: ES
- <10>: RBS30B-ATF1mut: ES
- <10*>: RBS30C-ATF1mut: ES
- <12>: ATF1mutB-Term: XP
- <13>: ATF1mutc-Term: XP
Take 2 ligations
transformations (@ 2:30 pm)
- prepare 9 plates, review the resistance of backbones
- use fresh Top10 cells, pUC19 control, 200 μL
