Daniel:Notebook/HiResChrPaint/2014-8-20

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Padlock Probes (Started 8/13/2014)[edit]

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Lambda Exonuclease Digestion[edit]

Since it looks like I might have less DNA than I originally thought (makes sense, several hundred ug is pretty unlikely), I'm going to run the lambda exonuclease digestion on the remaining padlock probes, A and B.

Reaction Table

Reagent PPRL-A (uL) PPRL-B (uL)
Amplicon 45 55
ug Input 20.7 25.6
Lambda Exo Buffer (10X) 10 10
Lambda Exonuclease (5 U/uL) 5 5
nf H2O 40 30
Total 100 100
  1. Set up reaction according to table
  2. Incubate at 37C for 2 hours
  3. Heat inactivate for 10 minutes at 75C
  4. Purify using ssDNA columns, elute with 20 uL nfH2O

Nanodrop Results[edit]

Sample ng/uL ug in 20 uL Input (ug) Efficiency (%) ug Total
PPRL-A 132.8 2.7 20.7 12.83
PPRL-B 214 4.3 25.6 16.72 6.9

This result is good enough to proceed with.

USER/DpnII Digestion[edit]

Reaction Table

Reagent PPRL (uL)
amplicon 30
ug Input 2.0
USER 5
nf H2O 45
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 PPRL (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

Nanodrop Results[edit]

Sample ng/uL ssDNA ug in 20 uL Total
PPRL-DpnII-A 38.7 0.8
PPRL-DpnII-B 35.3 0.7 1.5

Should be enough to label with.