Daniel:Notebook/HiResChrPaint/2014-8-19: Difference between revisions
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==Lambda Exonuclease digestion== | ==Lambda Exonuclease digestion== | ||
I'll just be using 10 uL of PPRL-A for this one, since it would appear that 10 uL is approximate 20 ug of probe. | |||
'''Reaction Table''' | '''Reaction 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="font-size:12pt;font-weight:bold" | |- style="font-size:12pt;font-weight:bold" | ||
|style="background-color:#C5D9F1" width=" | |style="background-color:#C5D9F1" width="112" height="15" valign="bottom" | Reagent | ||
|style="background-color:#FCD5B4" width="65" align="center" valign="bottom" | uL | |style="background-color:#FCD5B4" width="65" align="center" valign="bottom" | uL | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
|style="background-color:#C5D9F1" height="15" valign="bottom" | Amplicon | |style="background-color:#C5D9F1" height="15" valign="bottom" | Amplicon | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 10 | ||
|- style="font-size:12pt" | |||
|style="background-color:#C5D9F1" height="15" valign="bottom" | ug in 10 uL | |||
| align="center" align="center" valign="bottom" | 23.0 | |||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
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|- style="font-size:12pt" | |- style="font-size:12pt" | ||
|style="background-color:#C5D9F1" height=" | |style="background-color:#C5D9F1" height="30" valign="bottom" | Lambda Exonuclease (5 U/uL) | ||
| align="center" align="center" valign="bottom" | 5 | | align="center" align="center" valign="bottom" | 5 | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
|style="background-color:#C5D9F1" height="15" valign="bottom" | nf H2O | |style="background-color:#C5D9F1" height="15" valign="bottom" | nf H2O | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 75 | ||
|- style="font-size:12pt" | |- style="font-size:12pt" |
Revision as of 21:11, 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
I'll just be using 10 uL of PPRL-A for this one, since it would appear that 10 uL is approximate 20 ug of probe.
Reaction Table
Reagent | uL |
Amplicon | 10 |
ug in 10 uL | 23.0 |
Lambda Exonuclease Buffer (10X) | 10 |
Lambda Exonuclease (5 U/uL) | 5 |
nf H2O | 75 |
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