SBB09Ntbk-Patrick Harrigan

February 28, 2009

I made 100uM dillutions from my oligos and spun them down.
For a wobble reaction for KILLR, I added to a PCR tube:

 29 uL water
 5 uL Expand 10x Buffer 2
 5 uL 10x dNTPs (2 mM in each; 0.2 mM final conc)
 5 uL Oligo Oph22F (100uM)
 5 uL Oligo Oph23R (100uM)
 0.75 uL Expand Polymerase 1

For my PCR Product traA, First I diluted my Oligos

  9uL Water
  1uL 100uM Oph24F/R

Then I set up the following reaction in a PCR tube

<pre>
24uL ddH2O
3.3uL 10x Expand Buffer "2"
3.3uL dNTPs (2mM in each)
1uL Oligo 1, 10uM
1uL Oligo 2, 10uM
0.5uL Expand polymerase "1"
0.5uL Template DNA

For KILLR I ran the thermocycler on the wobble program. For traA, the thermocycler was run at 4k55

February 23 , 2009

traA

I added 2 uL from my PCR reaction and a 0.25 uL of loading dye and ran an analytic gel: 0. ladder 1. susan 2. Josh 3. Kamran 4. Roger 5. Patrick 8. roger 9. 10. sadao 11. carlos
My product is in the fith lane, I am not sure why there are two fragments.
I cleaned up my traA PCR reaction using the following protocol:

#Add 100 uL of Zymo ADB buffer (brown bottle) to the reaction.
#Transfer into the Zymo column (small clear guys)
#Add 500uL of Ethanol and pipette up and down to mix
#spin through, discard waste.
#Add 200 uL of PE or Zymo Wash buffer (which is basically 70% ethanol)
#spin through, discard waste.
#Add 200 uL of PE or Zymo Wash buffer
#spin through, discard waste.
#spin for 90 seconds, full speed to dry.
#elute with water into a fresh Eppendorf tube

I did not dry for long enough and may have gotten insufficient elution. I eluted with 50uL instead of 33 to try to make sure all DNA was eluted.

KILLR

For my wobble products, I digested the entire extension reaction-worth of DNA.

*I Set up the following reaction in a PCR tube:
  50uL eluted DNA
  5.7uL NEB Buffer 2
  1uL EcoRI
  1uL BamHI
*I mixed thoroughly by slamming the tube upside down on the table, and then gave it a quick spin to move the liquid to the bottom of the tube.
*I incubated the reaction at 37 degrees on the thermocycler

After digesting, I did a small Zymo cleanup:

#Add 100 uL of Zymo ADB buffer (brown bottle) to the reaction.
#Transfer into the Zymo column (small clear guys)
#Add 500uL of Ethanol and pipette up and down to mix
#spin through, discard waste.
#Add 200 uL of PE or Zymo Wash buffer (which is basically 70% ethanol)
#spin through, discard waste.
#Add 200 uL of PE or Zymo Wash buffer
#spin through, discard waste.
#spin for 90 seconds, full speed to dry.
#elute with water into a fresh Eppendorf tube