Daniel:Notebook/HiResChrPaint/2014-8-19: Difference between revisions
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>Djacobse (Created page with "=Padlock Probes (Started 8/13/2014)= Back to Calendar ==Ethanol Precipitation ([[Daniel:Notebo...") |
>Djacobse |
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===Nanodrop Results=== | ===Nanodrop Results=== | ||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#CCC0DA;font-size:12pt" align="center" valign="bottom" | |||
| width="65" height="15" | Sample | |||
| width="65" | ng/uL (1:5) | |||
| width="65" | ng/uL | |||
| width="65" | ug in 60 uL | |||
| width="65" | ug total | |||
|- style="font-size:12pt" | |||
|style="background-color:#D9D9D9" height="15" valign="bottom" | PPRL-A | |||
| align="center" align="center" valign="bottom" | 459.9 | |||
| align="center" align="center" valign="bottom" | 2,299.5 | |||
| align="center" align="center" valign="bottom" | 138.0 | |||
| align="center" align="center" valign="bottom" | | |||
|- style="font-size:12pt" | |||
|style="background-color:#D9D9D9" height="15" valign="bottom" | PPRL-B | |||
| align="center" align="center" valign="bottom" | 465.9 | |||
| align="center" align="center" valign="bottom" | 2,329.5 | |||
| align="center" align="center" valign="bottom" | 139.8 | |||
|style="font-weight:bold" align="center" align="center" valign="bottom" | 277.7 | |||
|} | |||
This seems to be a pretty ridiculous amount of probe, but it is consistent with my original nanodrop reading (4822.2 ng/uL of a 20 uL sample, undiluted). I'll push ahead cautiously with a large amount of the probe (but not all). | |||
==Lambda Exonuclease digestion== | ==Lambda Exonuclease digestion== |
Revision as of 21:07, 19 August 2014
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
Sample | ng/uL (1:5) | ng/uL | ug in 60 uL | ug total |
PPRL-A | 459.9 | 2,299.5 | 138.0 | |
PPRL-B | 465.9 | 2,329.5 | 139.8 | 277.7 |
This seems to be a pretty ridiculous amount of probe, but it is consistent with my original nanodrop reading (4822.2 ng/uL of a 20 uL sample, undiluted). I'll push ahead cautiously with a large amount of the probe (but not all).
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