SBB09Ntbk-DougDensmore

Doug Densmore

I started my first PCR today. Plus I started my notebook. Here are the protocols I need to follow for PCR.

1.Make an oligo dilution of (after reconstituting the oligo):

 9uL Water
 1uL 100uM oligo

Now I have a 10uM oligo.

2. Set up the following reaction in a PCR tube:

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

I did this for both my ChaI and Inv parts.

3. Next I ran my analytic gel with:

3uL of PCR product
1uL of loading buffer

I used 5uL of the ladder.

After examining the gels, I found that the Inv did not work. Therefore, I re-did the PCR for Inv. However I was able to continue with ChaI.

4. Regular Zymo Cleanup

1. Add 180 uL of Zymo ADB buffer (brown bottle) to a 33uL or 50uL reaction.
2. Transfer into the Zymo column (small clear guys)
3. spin through, discard waste.
4. Add 200 uL of PE or Zymo Wash buffer (which is basically 70% ethanol)
5. spin through, discard waste.
6. Add 200 uL of PE or Zymo Wash buffer
7. spin through, discard waste.
8. spin for 90 seconds, full speed to dry.
9. elute with water into a fresh Eppendorf tube, use the same volume of water as the volume of the original reaction

This process went fine so I then continued with the digest.

5. EcoRI/BamHI Digest

• Set up the following reaction:

 8uL of eluted PCR product
 1uL of NEB Buffer 2
 0.5uL EcoRI
 0.5uL BamHI

• 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

Finally I did a gel purify.

6. Zymo Gel Purify

1. cut out bands minimizing extra gel matter.
2. put in ependorf tube and add 600uL of Zymo ADB buffer (brown bottle).
3. heat at 55, shake and/or vortex until the gel has dissolved.
4. If the DNA is <300bp add 250uL of isopropanol
5. transfer into the Zymo column inside a collection tube (small clear guys)
6. spin through, discard waste.
7. Add 200 uL of PE buffer (which is basically 70% ethanol)
8. spin through, discard waste.
9. Add 200 uL of PE buffer
10. spin through, discard waste.
11. spin for 90 seconds, full speed to dry.
12. elute with 8.5 uL of water into a fresh Eppendorf tube

This was all I had time for.

Douglas Densmore 18:52, 2 March 2009 (EST)

Today I came back from my trip to Seattle and picked up where I left off.

Therefore I had to both begin with my two PCR attempts with Inv (W) and (D) as well as start with my ChaI digest.

Starting with the ChaI digest I did a ligation:

7. 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

After this I transformed this:

8. 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
  3. Add 30 uL of KCM salts
  4. Put your ligation mixture on ice, let it cool a minute or two
  5. Add 75 uL of the cell cocktail to the ligation, pipette up and down gently to mix
  6. Let sit on ice for 10 min
  7. Heat shock for 2 min at 42
  8. Put back on ice for 1 min
  9. For ampicillin selection, you can plate immediately, otherwise:
 10. Add 100uL of LB, let shake in the 37 degree incubator for 40 min
 11. Plate on selective antibiotics, let incubate overnight

I was able to skip steps 1-4 since the mixture was made. At this point I was able to stop with ChaI for the day.

The second piece was for me to get at least to the Zymo Clean up of the two PCR reactions for Inv. I was able to do this. I will have to go into the lab tomorrow to transform this.