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User:University of Washington/8 July 2008
From 2008.igem.org
Contents |
BioBrick Promoter Construct Sequencing
- Because the previous forward sequencing reaction and analysis yielded high-noise results, the forward sequencing protocol was carried out a second time.
- VF2 primer (100pmol/uL) was diluted 1 to 100 with distilled water.
- 8 uL of diluted VF2 primer (1pmol/uL) was added to four Eppendorf tubes.
- 4 uL of miniprepped plasmid DNA for parts I20260, I20268, I20269, and I20270 were added to tubes of primer, after appropriate labeling of the tubes.
- Plasmid template and VF2 primer solutions were resubmitted to the UW DNA Sequencing Facility for reaction and analysis.
- The index and middle fingers were crossed.
RP4 Conjugation
·Testing DH5α and S17-1+RP4 resistance to Chloremphenicol and Ampicillin.
·Testing S17-1 to DH5α conjugation using RP4 plasmid and conjugation protocol #1.
·Make glycerol stock of S17-1+RP4
Non-RP4 Conjugation
·Plate out CV13(Yep13) and pDPT51
LuxR from pLac
- Made glycerol stocks of R0010 and I763004.
- Miniprepped and sent in for sequencing part I763004.
LuxR from AraC and TetR
- Cells with AraC plasmid grew on Amp plate and was stored in the fridge. (Note: The cells were spread by glass rod instead of scraping using the metal rod, so single colonies can be found only on the side of the plate)
- Miniprepped 5 cultures of AraC.
- Ran gel (5 ul DNA + 2 ul dye) and found that five plasmid from 5 tubes have the same length. The plasmid was then combined into one tube.
- Nanodropped AraC plasmid: 201.6 ng/ml; 260/280 = 1.89; 260/230 = 2.17
- Performed the first part of QuikChange Mutagenesis
- Dilute Primers(100 pmole/ul) 1:10 ==> 10pmole/ul with deionized water.
- Mix(2 reactions in 2 PRC tubes))
| Materials | Reaction#1 | Reaction#2 |
|---|---|---|
| AraC plasmid | 39.3 ul | 39.5 ul |
| 10X pfuTurbo reaction buffer | 5 ul | 5 ul |
| dNTP mix | 1 ul | 1 ul |
| primer AraC-F | 0.806 ul AraC1F | 0.728 ul AraC2F |
| primer AraC-R | 0.806 ul AraC1R | 0.728 ul AraC2R |
| DMSO | 3 ul | 3 ul |
- add 1 ul pfuTurbo in both
- Temperature Cycle(There are small changes from our protocol page.
| Segment | Cycles | Temperature | Time |
|---|---|---|---|
| 1 | 1 | 95°C | 2 minute |
| 2 | 18 | 95°C | 50 seconds |
| 60°C | 50 seconds | ||
| 68°C | 3 mins | ||
| 3 | 1 | 68°C | 7 minutes |
| let it sit in 4°C overnight |
Lambda Red Recombineering of RP4 (Bryan)
Ordered pKD76 helper plasmid from Yale CGSC, containing Lambda Red recombinatory genes and a CamR cassette.
Contacted Don Court at Center for Cancer Research for Lambda Red recombineering advice. Received protocols.
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