Lab Notebook - John

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iGem 2009
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7-29-09

Bgl/BamHI Digest of vector plasmid

• Set up the following reaction:

 3uL of vector DNA
 3uL of NEB Buffer 2
 1.5uL Bgl
 1.5uL BamHI
 21ul of ddH2O

• Incubate at 37 degrees on the thermocycler for 1hr
• Run an agarose gel, and melt with 600uL ADB buffer at 55 degrees. ****NOTE: If you are running short of time, this is an acceptable stopping point

Ligation of EcoRI/BamHI digests

• Set up the following reaction:

 6.5uL ddH2O
 1uL T4 DNA Ligase Buffer (small red or black-striped tubes)
 1uL Vector digest
 1uL Insert digest
 0.5uL T4 DNA Ligase

• Pound upside down on the bench to mix
• Give it a quick spin to send it back to the bottom of the tube
• Incubate on the benchtop for 30min
• Put on ice and proceed to the transformation

Transformation by heat-shock
Competent cells are stored as 200uL aliquots in the -80 freezer as a communal stock.

  1. Thaw a 200 uL aliquot of cells on ice
  2. Add 50 uL of water to the cells
  3. Add 30 uL of KCM to the cells 
  4. Put your ligation mixture on ice, let cool a minute or two
  5. Add 70 uL of the cell cocktail to the ligation, stir to mix
  6. Let sit on ice for 10 min
  7. Heat shock for 90 seconds at 42 (longer incubation may work better)
  8. Put back on ice for 1 min
  9. Add 100uL of 2YT, let shake in the 37 degree incubator for 1 hour
 10. Plate 70+ uL on selective antibiotics, let incubate at 37 degrees overnight

7-3-09

AC ---> competent died on kan. Spec contamination
CA ---> competent died on kan and spec

CK ---> competent died on amp and spec
KC ---> competent died on amp and spec

AK ---> competent died on cam and spec
KA ---> competent died on cam and spec

Pir lefty - competent - did not grow on kan amp or cam. grew on spec
Pir righty - competent - did not grow on kan amp or cam. grew on spec

6-25-09

It was annoying me that there was no fully written out work flow for the SOEing reaction as well as some missing critical details so I wrote up a full run: full SOEing.

6-15-09

Over the weekend, I ended up fishing out the Tev sequence. There are multiple types of Tev to try. I was unable to post it on the parts page though since there is no space left to add and only Chris can edit.

6-11-09

Today I recovered from running that reaction. I also made this site pretty.

6-10-09

Today I ran a reaction.