IGEM:MIT/2006/Notebook/2006-8-1: Difference between revisions
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==Run Gel of BAT2 and THI3 PCR Products== | ==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 | Nothing showed (including + control)- so we will PCR BAT2 and THI3 again with 1 μL yeast genomic dna | ||
==PCR BAT2 and THI3 (2nd attempt)== | |||
*Used 1μL 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.== | ==Fax Dr. Schweizer for pUCP18 etc.== | ||
Line 20: | Line 23: | ||
#found 2 new fax numbers and successfully sent a fax to University of Calgary and Dr. Schweizer's updated office fax | #found 2 new fax numbers and successfully sent a fax to University of Calgary and Dr. Schweizer's updated office fax | ||
==Gel extract fragments== | |||
==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)== | |||
#RBS<sub>30A</sub>-CDS-Term: XP | #RBS<sub>30A</sub>-CDS-Term: XP | ||
#RBS<sub>30B</sub>-CDS-Term: XP | #RBS<sub>30B</sub>-CDS-Term: XP | ||
Line 30: | Line 41: | ||
#E0840: XP | #E0840: XP | ||
== | ==9 ligations using gel extracts 1-8 (7/31) above== | ||
#R0040+1->Prom-RBS<sub>30A</sub>-CDS-Term | #R0040+1->Prom-RBS<sub>30A</sub>-CDS-Term | ||
#R0040+2->Prom-RBS<sub>30B</sub>-CDS-Term | #R0040+2->Prom-RBS<sub>30B</sub>-CDS-Term | ||
Line 52: | Line 50: | ||
#7+Term->RBS<sub>30B</sub>-ATF1<sub>mut</sub>-Term | #7+Term->RBS<sub>30B</sub>-ATF1<sub>mut</sub>-Term | ||
#R0040+8(E0840)->CP-RBS-GFP-Term | #R0040+8(E0840)->CP-RBS-GFP-Term | ||
# | #osmY+2->osmY-RBS<sub>30B</sub>-CDS-Term | ||
==Take 2 Digestions (same as 7/31)== | |||
#note number system! | |||
#*<1>: RBS<sub>30A</sub>-CDS-Term: XP ***this got really messed up because the number 1 looks like the number 7*** | |||
#*<2>: RBS<sub>30B</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 | |||
#*<13>: ATF1mut<sub>c</sub>-Term: XP | |||
*Order of lanes: 2, 3, 4, RBS<sub>32C</sub>-ATF1mut: ES, 9, 10 | |||
[[Image:8-1gel1.jpg]] | |||
*Order of lanes: 10***, 12, 13, 14 | |||
[[Image:8-1gel2.jpg]] | |||
==13 ''Take 2'' ligations== | |||
#R0040 + <1> ***BAD...the "1" was really "7"; didn't transform this*** | |||
#R0040 + <2> | |||
#R0040 + <3> | |||
#R0040 + <4> | |||
#osmY + <1> ***BAD...the "1" was really "7"; didn't transform this*** | |||
#osmY + <4> | |||
#<9> + B0015 | |||
#<10> + B0015 | |||
#<10***> + B0015 | |||
#B0030 + <12> | |||
#B0030 + <13> | |||
#B0032 + <12> | |||
#B0032 + <13> | |||
==transformations (@ | ==transformations (@ 5:30 pm)== | ||
#prepare | #prepare 23 plates, review the resistance of backbones | ||
#*everything in B0015 backbone is Amp/Kan!! rest are just Amp | |||
#use fresh Top10 cells, pUC19 control, 200 μL | #use fresh Top10 cells, pUC19 control, 200 μL |
Latest revision as of 15:30, 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
PCR BAT2 and THI3 (2nd attempt)
- Used 1μL 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.
- 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 ***this got really messed up because the number 1 looks like the number 7***
- <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
- Order of lanes: 2, 3, 4, RBS32C-ATF1mut: ES, 9, 10
- Order of lanes: 10***, 12, 13, 14
13 Take 2 ligations
- R0040 + <1> ***BAD...the "1" was really "7"; didn't transform this***
- R0040 + <2>
- R0040 + <3>
- R0040 + <4>
- osmY + <1> ***BAD...the "1" was really "7"; didn't transform this***
- osmY + <4>
- <9> + B0015
- <10> + B0015
- <10***> + B0015
- B0030 + <12>
- B0030 + <13>
- B0032 + <12>
- B0032 + <13>
transformations (@ 5:30 pm)
- prepare 23 plates, review the resistance of backbones
- everything in B0015 backbone is Amp/Kan!! rest are just Amp
- use fresh Top10 cells, pUC19 control, 200 μL