Daniel:Notebook/HiResChrPaint/2014-8-18

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Revision as of 15:24, 18 August 2014 by >Djacobse
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Padlock Probes (Started 8/13/2014)

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qPCR

I need more padlock probes, so I'm going to repeat qPCR with 24x samples.

Reaction Table

Component 1X Reaction 24.5X Scaled
Padlock Probes 0.2 4.9
100 uM pAP1V41U 0.4 9.8
100 uM pAP2V4 0.4 9.8
KAPA SYBR Fast Master Mix (2X) 50 1225
H2O 49 1200.5
Total 100 2450
  1. Prepare master mix according to table above
  2. Add 100 uL reaction to PCR tubes (5 total)
  3. qPCR Protocol
    1. 95C 30 sec
    2. 95C 10 sec
    3. 55C 20 sec
    4. 72C 30 sec
    5. goto (3.2) 17 times
    6. 72C 2 min
    7. Hold at 16C forever
  4. Ethanol Precipitation

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