Daniel:Notebook/HiResChrPaint/2014-8-20: Difference between revisions
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'''DpnII Primer Table''' | '''DpnII Primer Table''' | ||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | ||
|- style="background-color:#B8CCE4;font-size:12pt" | |- style="background-color:#B8CCE4;font-size:12pt;font-weight:bold" align="center" | ||
| width=" | | width="93" height="31" | Reagent | ||
| width="65" | | width="65" | PPRL-A (uL) | ||
| width="65" | PPRL-B (uL) | |||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
|style="background-color:#FCD5B4" height="15" valign="bottom" | 10X DpnII Buffer | |style="background-color:#FCD5B4" height="15" valign="bottom" | 10X DpnII Buffer | ||
| align="center" align="center" valign="bottom" | 10 | |||
| align="center" align="center" valign="bottom" | 10 | | align="center" align="center" valign="bottom" | 10 | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
|style="background-color:#FCD5B4" height="30" valign="bottom" | 100 uM RE-DpnII-V4 guide oligo | |style="background-color:#FCD5B4" height="30" valign="bottom" | 100 uM RE-DpnII-V4 guide oligo | ||
| align="center" align="center" valign="bottom" | 5 | |||
| align="center" align="center" valign="bottom" | 5 | | align="center" align="center" valign="bottom" | 5 | ||
|- style="font-size:12pt;font-weight:bold" | |- style="font-size:12pt;font-weight:bold" | ||
|style="background-color:#FCD5B4" height="15" valign="bottom" | Total | |style="background-color:#FCD5B4" height="15" valign="bottom" | Total | ||
| align="center" align="center" valign="bottom" | 15 | |||
| align="center" align="center" valign="bottom" | 15 | | align="center" align="center" valign="bottom" | 15 | ||
Revision as of 18:12, 20 August 2014
Padlock Probes (Started 8/13/2014)
Lambda Exonuclease Digestion
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
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
Reaction Table
Reagent | PPRL-A (uL) | PPRL-B (uL) |
amplicon | 15 | 15 |
ug Input | 2.0 | 3.2 |
USER | 5 | 5 |
nf H2O | 60 | 60 |
Total | 80 | 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-A (uL) | PPRL-B (uL) |
10X DpnII Buffer | 10 | 10 |
100 uM RE-DpnII-V4 guide oligo | 5 | 5 |
Total | 15 | 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