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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
|
- Set up reaction according to table
- Incubate at 37C for 2 hours
- Heat inactivate for 10 minutes at 75C
- 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
|
- 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
|
PPRL (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
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.