IGEM:Harvard/2006/Perry projects: Difference between revisions
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**X ul midiprep for ~1ug DNA, 2.5ul Buffer2, 0.5ul SpeI/XbaI, 0.5ul PstI, 0.25ul BSA(100X), Y ul water for total volume of 25ul | **X ul midiprep for ~1ug DNA, 2.5ul Buffer2, 0.5ul SpeI/XbaI, 0.5ul PstI, 0.25ul BSA(100X), Y ul water for total volume of 25ul | ||
*** Y should equal 21.25 minus X. Don't worry about having exactly 0.25ul BSA; just convince yourself there's a little bit of BSA on the end of your pipet tip, and that it's entering your mixture. | *** Y should equal 21.25 minus X. Don't worry about having exactly 0.25ul BSA; just convince yourself there's a little bit of BSA on the end of your pipet tip, and that it's entering your mixture. | ||
**Incubate at 37dC overnight (12h or so), | **Incubate at 37dC overnight (12h or so), 80dC for 20min, 4dC forever. There should be a program called "Digest" on either of the two grey PCR machines; just change the 37dC step to whatever time. | ||
*Prepare three PCRs using "S/C/E no mut in pET" minipreps and StrepSCDF/StrepSCDRS(5uM) primers in "S,C,E" box, and PCR Supermix in "Primers/Stuff" box. | *Prepare three PCRs using "S/C/E no mut in pET" minipreps and StrepSCDF/StrepSCDRS(5uM) primers in "S,C,E" box, and PCR Supermix in "Primers/Stuff" box. | ||
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**" + " + " + 1ul "C, no mut in pET" miniprep | **" + " + " + 1ul "C, no mut in pET" miniprep | ||
**" + " + " + 1ul "E, no mut in pET" miniprep | **" + " + " + 1ul "E, no mut in pET" miniprep | ||
***Incubate using "PCR PT" program on either grey PCR machine. | |||
===Tuesday=== | ===Tuesday=== | ||
*Treat the digests with alkaline phosphatase (CIP). | *Treat the digests with alkaline phosphatase (CIP). To each of the 25ul digests, add 1ul alkaline phosphatase and 3ul 10X phosphatase buffer. Incubate at 37dC for one hour, 4dC forever. | ||
*While you're waiting, pour a 1% 100ml agarose gel, and also perform minipreps of R0010 and B0032 liquid cultures from yesterday. |
Revision as of 06:48, 7 August 2006
BioBricks assembly
The final constructs:
- R0010-B0032-Lpp29-OmpA66-Streptavidin
- R0010-B0032-Lpp29-OmpA159-Streptavidin
The parts
R0010 is a Lac promoter, B0032 is a ribosome binding site (medium strength, same one used in the GFP expression device), Lpp29-OmpA66 and Lpp29-OmpA159 are two versions of the membrane display scaffold.
There are several different versions of streptavidin that may be used. We have BioBricks of strepavidin clones from the Ting lab: wild-type, wild-type + His6tag, and dead, which I refer to as StrepW, StrepH, and StrepD.
Available sequenced and midiprepped parts
In Lpp-OmpA box...
- Lpp29, OmpA66, OmpA159 in pCR-2.1 Topo vector
- Lpp29, OmpA66, OmpA159 in pSB1A2 vector
- Lpp29-OmpA66 in pSB1A2
In StrepW,H,D box...
- StrepW, StrepH, StrepD in pCR-2.1 Topo vector
Available digested and gel purified parts, ready for ligation
- R0010, S/P (CIPed)
- Lpp29, E/S
- Lpp29, S/P (CIPed)
- OmpA66, X/P
- OmpA159, X/P
- Lpp29-OmpA66, S/P (CIPed)
- StrepW, StrepH, StrepD, X/P
- pSB1A2, E/P ("1")
- pSB1A2, E/S ("2")
- pSB1A2, X/P ("3")
BioBricks PCR
We're working on getting BioBricks of single-chain dimeric streptavidin clones from the Aslan lab: SCD-NM (single-chain dimer, not mutant), C2, and E2 (two mutant clones), which I refer to as S, C, and E. I just received a core streptavidin (streptavidin has a full-length version and a functional truncated core version) from the Dr. Takeshi Sano, called pTSA13, which I refer to as pTSA13 or just 13.
There are also two clones of the Lpp-OmpA scaffold which I received recently from the Georgiou lab, 481 (Lpp1-9-OmpA46-66-Bla) and 488 (Lpp1-9-OmpA46-145-Bla).
Goals for week of 8/6/06
Assembly
- Lpp29-OmpA159
- Lpp29-OmpA66-StrepW, H, or D
- R0010-B0032
BioBrick PCR
- SCD-NM, C2, or E2 clones of single-chain dimeric streptavidin from Aslan lab, with PstI site removed by mutagenesis
- pTSA13 streptavidin clone from Sano lab
- 481 or 488 construct of Lpp-OmpA from Georgiou lab
Agenda
Monday
- Inoculate liquid cultures, 3ml LB + 30ul amp(5mg/ml), of R0010 and B0032. You can use a R0010 streak on a carb plate labeled 7/31/06 in the B2 refrigerator, and a B0032 streak on a carb plate labeled 8/2/06. Make sure you start incubating/shaking them late enough in the day that you'll be around in 16 hours the following day to perform the miniprep.
- Prepare 25ul double digests using midipreps of Lpp29 (SpeI/PstI), OmpA159 (XbaI/PstI), and Lpp29-OmpA66 (SpeI/PstI) in pSB1A2 (Lpp-OmpA box). You should aim for around 1ug of DNA in each digest.
- X ul midiprep for ~1ug DNA, 2.5ul Buffer2, 0.5ul SpeI/XbaI, 0.5ul PstI, 0.25ul BSA(100X), Y ul water for total volume of 25ul
- Y should equal 21.25 minus X. Don't worry about having exactly 0.25ul BSA; just convince yourself there's a little bit of BSA on the end of your pipet tip, and that it's entering your mixture.
- Incubate at 37dC overnight (12h or so), 80dC for 20min, 4dC forever. There should be a program called "Digest" on either of the two grey PCR machines; just change the 37dC step to whatever time.
- X ul midiprep for ~1ug DNA, 2.5ul Buffer2, 0.5ul SpeI/XbaI, 0.5ul PstI, 0.25ul BSA(100X), Y ul water for total volume of 25ul
- Prepare three PCRs using "S/C/E no mut in pET" minipreps and StrepSCDF/StrepSCDRS(5uM) primers in "S,C,E" box, and PCR Supermix in "Primers/Stuff" box.
- 45ul PCR Supermix + 2ul StrepSCDF(5uM) + 2ul StrepSCDRS(5uM) + 1ul "S, no mut in pET" miniprep
- " + " + " + 1ul "C, no mut in pET" miniprep
- " + " + " + 1ul "E, no mut in pET" miniprep
- Incubate using "PCR PT" program on either grey PCR machine.
Tuesday
- Treat the digests with alkaline phosphatase (CIP). To each of the 25ul digests, add 1ul alkaline phosphatase and 3ul 10X phosphatase buffer. Incubate at 37dC for one hour, 4dC forever.
- While you're waiting, pour a 1% 100ml agarose gel, and also perform minipreps of R0010 and B0032 liquid cultures from yesterday.