Daniel:Notebook/HiResChrPaint/2014-8-19

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Revision as of 20:19, 19 August 2014 by >Djacobse (Created page with "=Padlock Probes (Started 8/13/2014)= Back to Calendar ==Ethanol Precipitation ([[Daniel:Notebo...")
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Padlock Probes (Started 8/13/2014)

Back to Calendar

Ethanol Precipitation (From 8/18/2014)

  1. Remove from -80C centrifuge at 4400 rpm for 30 minutes at 4C
  2. Add 700 uL 75% EtOH
    1. Break up the pellet and transfer to a 1.5mL tube
  3. Centrifuge once more at 14000 rpm for 10 minutes
  4. Take out supernatant and dry in laminar flow hood for 20 minutes
  5. Resuspend in 20 uL nf H2O

Nanodrop Results

Lambda Exonuclease digestion

Reaction Table

Reagent uL
Amplicon 30
Lambda Exonuclease Buffer (10X) 10
Lambda Exonuclease 5
nf H2O 55
Total 100
  1. Set up reaction according to table
  2. Incubate at 37C for 1 hour
  3. Purify using 2 ssDNA columns, elute with 20 uL nfH2O each

USER/DpnII Digestion

Reaction Table

Reagent uL
amplicon 35
USER 5
nf H2O 40
Total 80
  1. Set up reaction according to table in a 0.2 mL PCR tube
  2. Incubate for 2 hours at 37C
  3. Add 15 uL according to DpnII Primer table (below)

DpnII Primer Table

Reagent uL
10X DpnII Buffer 10
100 uM RE-DpnII-V4 guide oligo 5
Total 15
  1. Incubate at 94C for 2 minutes
  2. Cool to 37C and incubate for 3 minutes
  3. Add 5 uL DpnII and incubate at 37C for 2 hours
  4. Heat inactivate DpnII at 65C for 20 minutes