SBB11Ntbk-Keith Licardo

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Keith Licardo 14:02, 15 February 2011 (EST)

sbb1103 part PCR
Expand PCR with oligos ss28f and ss28r on MG1655 genome.
The oligos were already diluted at 10 uM and so I used them provided oligos directly.
Followed the Expand PCR protocols as listed in Spring 140L website. Used Expand Buffer.
The part was around 700 bp long so I decided to use 2K55 for the thermocycler algorithm.

Keith Licardo 14:01, 17 February 2011 (EST)

sbb1103 PCR Analytical Gel
Set-up into 0.5 mL tube: 5 uL of loading dye and only 2 uL of PCR product. Someone else did the gel upstairs.
Lane 11 was mine. The fragment in that lane matched the 700-kb fragment on the very first lane. This indicates that I was able to take out the right fragment.

The rest of the PCR product was then put through zymo clean up using the protocol from the BE140L site. I left the DNA fragments in the regular zymo filter for around 15 minutes as I had to go upstairs to do the preparative gel (gel purification) for Bth8112. The DNA might be damaged if it dried up in the zymo filter. I followed the rest of the protocol and washed the fragments twice with the zymo wash buffer and dried it up with the centrifuge for 90 secs.

Keith Licardo 14:16, 17 February 2011 (EST)

cut-and-paste from vector to another for Bth8112

Digesting out Bth8112
Cutting Bth8112 out from vector pBca9145.
Followed the protocol for "EcoRI/BamHI Digest of PCR Products." Used 0.5 uL of the pBca9145-Bth8112. Added everything to the original tube for pBca9145-Bth8112. Then added the following: 3 uL of DDW, 1 uL of NEB buffer, 0.5 uL of EcoRI, 0.5 uL of BamHI. Spun with the mini spin after adding each. Pipetted all of the 10 uL from the original into a PCR tube but had to pipet out the rest at 0.5 uL three times. The PCR tube was then mini-spun and incubated for an hour.
The following is gel picture for the gel purification step.
Lane 5 was mine. Although the lane shows two blurry bands, the bands became more vivid with more time running the gel. Professor Anderson cut mine for me. The smaller fragment was put into a tube with 600 uL of ADB buffer and left in the 55 deg Celsius bath.

Keith Licardo 16:26, 18 February 2011 (EST)

cut-and-paste from vector to another for part Bth8112

Zymo Gel Purification of Bth8112
Followed the protocol for the Zymo gel purification of Bth8112 from yesterday. (Note: Sung-won mentioned that leaving the fragments in the gel might have been a better stopping point than leaving the fragments in the gel and ADB buffer.) The professor cut the gel for me, and no isopropanol was added as my fragment was larger than 300 bp. After the ADB buffer and liquified gel was filtered out, the fragments was then washed twice with 200 uL AW wash. The fragments was then dryed out for 90 seconds in the centrifuge. Finally, the fragments were eluted out with 8.5 uL of DDW.