SBB11Ntbk-MaryWang: Difference between revisions
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==[[User:Mary Wang|Mary Wang]] 13:38, 3 March 2011 (EST)== | |||
The plate with ffgfp did not grow. The plate with sbb1139 PGadA showed so a Miniprep was done | |||
==[[User:Mary Wang|Mary Wang]] 13:38, 1 March 2011 (EST)== | ==[[User:Mary Wang|Mary Wang]] 13:38, 1 March 2011 (EST)== | ||
Ligation of EcoRI/BamHI | Ligation of EcoRI/BamHI | ||
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6.5uL ddH2O, 1uL T4 DNA Ligase buffer, 1uL 1139 vector, 1uL 1601KC, T4 DNA Ligase | 6.5uL ddH2O, 1uL T4 DNA Ligase buffer, 1uL 1139 vector, 1uL 1601KC, T4 DNA Ligase | ||
==[[User:Mary Wang|Mary Wang]] 13:20, 24 February 2011 (EST)== | ==[[User:Mary Wang|Mary Wang]] 13:20, 24 February 2011 (EST)== | ||
Revision as of 15:14, 4 March 2011
Mary Wang 13:38, 3 March 2011 (EST)
The plate with ffgfp did not grow. The plate with sbb1139 PGadA showed so a Miniprep was done
Mary Wang 13:38, 1 March 2011 (EST)
Ligation of EcoRI/BamHI
6.5uL ddH2O, 1uL T4 DNA Ligase buffer, 1uL 2296 vector, 1uL 1601CA, T4 DNA Ligase
6.5uL ddH2O, 1uL T4 DNA Ligase buffer, 1uL 1139 vector, 1uL 1601KC, T4 DNA Ligase
Mary Wang 13:20, 24 February 2011 (EST)
EcoRI/BamHI Digest on pBjh1601KA-Bjh2296
Run Prep Gel
Zymo Gel Clean Up
elute with 8uL ddH2O
Mary Wang 13:20, 23 February 2011 (EST)
EcoRI, BamHI digest on PCR ss45f/ss45r on MG1655 gen. Prep gel on PCR. Showed up on analytical gel 4- lane 1
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- labeled MW9
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
