SBB11Ntbk-MaryWang: Difference between revisions
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==[[User:Mary Wang|Mary Wang]] 22:47, | ==[[User:Mary Wang|Mary Wang]] 22:47, 18 February 2011 (EST)== | ||
Gel zymo purification done on the digest of pBjh1601KA- labeled MW8 | Gel zymo purification done on the digest of pBjh1601KA- labeled MW8 | ||
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180uL of ADB buffer, 2X 200uL PE buffer- elute with 31uL of ddH2O (2uL used for PCR) | 180uL of ADB buffer, 2X 200uL PE buffer- elute with 31uL of ddH2O (2uL used for PCR) | ||
==[[User:Mary Wang|Mary Wang]] 14:18, 17 February 2011 (EST)== | ==[[User:Mary Wang|Mary Wang]] 14:18, 17 February 2011 (EST)== |
Revision as of 20:48, 19 February 2011
Mary Wang 22:47, 18 February 2011 (EST)
Gel zymo purification done on the digest of pBjh1601KA- labeled MW8
2X 200uL of PE buffer. Eluted with 8uL of ddH2O.
Analytical gel run on PCR on part sbb1139
Analytical Gel1 Lane 3 and 4 (Lane 3 is no DMSO, Lane 4 has DMSO)
DMSO PCR did not show up in Lane 4
Regular Zymo cleanup done
180uL of ADB buffer, 2X 200uL PE buffer- elute with 31uL of ddH2O (2uL used for PCR)
Mary Wang 14:18, 17 February 2011 (EST)
Analytical gel run on part ssb1139: Used 2uL product from previous day and 5uL dye. Analytical gel shows that the part was done incorrectly. Analytical Gel2- lane 5.
REdone (x2)
One: 24uL ddH2O, 3.3uL 10X expand buffer, 3.3 uL dNTP, 1 uL ss45r, 1uL ss45f, 0.5uL template DNA, 0.5 expand polymerase 1
Two: 21.7uL ddH2O, 3.3uL DMSO, 3.3uL 10X expand buffer, 3.3uL dNTP, 1uLss45r, 1uL ss45f, 0.5uL template DNA, 0.5 expand polymerase 1
Run at 2k45 instead of 2k55
Digest pBjh1601KA-Bjh2296 using: 8uL of eluted PCR product, 1uL of NEB Buffer 2, 0.5uL EcoRI and 0.5uL BamHI. Prep Gel run- shown on PrepGel2- lane 4. Cut out ~837 part- melted with 600uL ADB buffer, melt at 55C for 10 minutes then frozen.
Mary Wang 14:16, 15 February 2011 (EST)
PCR ss45r/ss45f on MG1655 genome.
Used Expand polymerase because it was a genome.
Put template DNA into PCR tube first then the Expand polymerase second.
Did not dilute the oligos.
Used cloning by PCR- basic PCR method.
Amounts used: 24uL ddH2O, 3.3uL 10X expand buffer, 3.3uL dNTP, 1uL Oligo 1, 1uL Oligo 2, 0.5 Expand polymerase 1, 0.5uL Template DNA
Run at 2k55