User:University of Washington/8 July 2008

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))
 * add 1 ul pfuTurbo in both
 * Temperature Cycle(There are small changes from our protocol page.

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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