Lab Notebook - John

Go Back iGem 2009 ProTips for Editing BootCamp

=Journal=

8-24-09
Plating the cotransformed iilk and mGFP on AKC. Also going to check the two 24 well block dilution series.

8-3-09
http://openwetware.org/wiki/Restriction_Digest_8-3

7-29-09
Bgl/BamHI Digest of vector plasmid

3uL of vector DNA 3uL of NEB Buffer 2 1.5uL Bgl 1.5uL BamHI 21ul of ddH2O Ligation of EcoRI/BamHI digests 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 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
 * Set up the following reaction:
 * 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
 * Set up the following reaction:
 * 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

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.