Daniel:Notebook/HiResChrPaint/2014-8-19
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Padlock Probes (Started 8/13/2014)
Ethanol Precipitation (From 8/18/2014)
- Remove from -80C centrifuge at 4400 rpm for 30 minutes at 4C
- Add 700 uL 75% EtOH
- Break up the pellet and transfer to a 1.5mL tube
- Centrifuge once more at 14000 rpm for 10 minutes
- Take out supernatant and dry in laminar flow hood for 20 minutes
- 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 |
- Set up reaction according to table
- Incubate at 37C for 1 hour
- 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 |
- Set up reaction according to table in a 0.2 mL PCR tube
- Incubate for 2 hours at 37C
- 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 |
- Incubate at 94C for 2 minutes
- Cool to 37C and incubate for 3 minutes
- Add 5 uL DpnII and incubate at 37C for 2 hours
- Heat inactivate DpnII at 65C for 20 minutes