Team:Brown/Notebook/July23

From 2010.igem.org

Friday, July 23, 2010

Observations on plates from yesterday

Lawns on amp plates with peking plasmid cells. Apparently, too many cells per plate.

Gel of miniprep ligations

Ran all 8 miniprep (WillRS+pGEM) ligations (lanes 2-8) with a 1kb ladder

Gel 72310.jpg

Imaging results: It appears we have the insert!

Next steps:

  • Digest with Nco1 and BamHI (BSA not important, but make sure to use the correct buffer and not too much DNA)
  • Gel-isolate ~800bp band
  • Prepare 2 samples for sequencing

Note - use NEB NcoI, BamHI, and Buffer3 only

Ran producs of 7/22 PCR

Of the 20ul PCR product, added 4ul loading buffer, placed 10 of 24ul mixture into each well, placed10ul ladder into each well. Image of gel will be tracked down

Digest of #1-4 pGEM WillRS constructs miniprep

Master mix (x4.5)

  • 58.75ul dH2O
  • 27ul 10x buffer 3
  • 6.75ul NcoI
  • 6.75ul BamHI
  • 2.7ul BSA

Reaction (50ul total volume)

  • 5ul NEB buffer 3 10x
  • 6ul DNA (miniprep)
  • 1.5ul NcoI
  • 1.5ul BamHI
  • 0.5ul BSA
  • 35.5ul dH2O

Started at 3:10pm

Retrieved from "http://2010.igem.org/Team:Brown/Notebook/July23"