Daniel:Notebook/HiResChrPaint/2014-8-19: Difference between revisions
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>Djacobse |
>Djacobse |
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#Incubate at 37C for 1 hour | #Incubate at 37C for 1 hour | ||
#Purify using 2 [[Daniel:Protocols/ssDNA Column|ssDNA columns]], elute with 20 uL nfH2O each | #Purify using 2 [[Daniel:Protocols/ssDNA Column|ssDNA columns]], elute with 20 uL nfH2O each | ||
==Gel Quantification Check== | |||
Based on the results above (no sample after Lambda digestion), I'm going to do a massive gel dilution series to see approximately what the concentration of PPRL-A is. I'll either repeat for B, or more likely assume it is on the same scale. | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#E6B8B7;font-size:12pt;font-weight:bold" align="center" | |||
| width="86" height="34" | Lane | |||
| width="86" | 2 (Ladder) | |||
| align="center" width="65" | 3 | |||
| align="center" width="65" | 4 | |||
| align="center" width="65" | 5 | |||
| align="center" width="65" | 6 | |||
| align="center" width="65" | 7 | |||
| align="center" width="65" | 8 | |||
| align="center" width="65" | 9 | |||
| align="center" width="65" | 10 | |||
|- style="font-size:12pt" | |||
|style="background-color:#C5D9F1" height="15" valign="bottom" | Dilution | |||
| align="center" align="center" valign="bottom" | 1 | |||
| align="center" align="center" valign="bottom" | 1 | |||
| align="center" align="center" valign="bottom" | 1 | |||
| align="center" align="center" valign="bottom" | 10 | |||
| align="center" align="center" valign="bottom" | 10 | |||
| align="center" align="center" valign="bottom" | 10 | |||
| align="center" align="center" valign="bottom" | 10 | |||
| align="center" align="center" valign="bottom" | 50 | |||
| align="center" align="center" valign="bottom" | 50 | |||
|- style="font-size:12pt" | |||
|style="background-color:#C5D9F1" height="15" valign="bottom" | uL Sample | |||
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 1 | |||
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 1 | |||
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 0.5 | |||
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 4 | |||
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 2 | |||
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 1 | |||
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 0.5 | |||
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 1 | |||
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 0.5 | |||
|- style="font-size:12pt" | |||
|style="background-color:#C5D9F1" height="15" valign="bottom" | uL TBE | |||
| align="center" align="center" valign="bottom" | 4.5 | |||
| align="center" align="center" valign="bottom" | 4.5 | |||
| align="center" align="center" valign="bottom" | 5 | |||
| align="center" align="center" valign="bottom" | 1.5 | |||
| align="center" align="center" valign="bottom" | 3.5 | |||
| align="center" align="center" valign="bottom" | 4.5 | |||
| align="center" align="center" valign="bottom" | 5 | |||
| align="center" align="center" valign="bottom" | 4.5 | |||
| align="center" align="center" valign="bottom" | 5 | |||
|- style="font-size:12pt" | |||
|style="background-color:#C5D9F1" height="15" valign="bottom" | uL buffer | |||
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 2 | |||
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 2 | |||
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 2 | |||
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 2 | |||
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 2 | |||
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 2 | |||
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 2 | |||
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 2 | |||
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 2 | |||
|- style="font-size:12pt" | |||
|style="background-color:#C5D9F1" height="15" valign="bottom" | Total | |||
| align="center" align="center" valign="bottom" | 7.5 | |||
| align="center" align="center" valign="bottom" | 7.5 | |||
| align="center" align="center" valign="bottom" | 7.5 | |||
| align="center" align="center" valign="bottom" | 7.5 | |||
| align="center" align="center" valign="bottom" | 7.5 | |||
| align="center" align="center" valign="bottom" | 7.5 | |||
| align="center" align="center" valign="bottom" | 7.5 | |||
| align="center" align="center" valign="bottom" | 7.5 | |||
| align="center" align="center" valign="bottom" | 7.5 | |||
|- style="font-size:12pt" | |||
|style="background-color:#C5D9F1" height="15" valign="bottom" | uL Added | |||
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 7.2 | |||
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 7.2 | |||
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 7.2 | |||
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 7.2 | |||
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 7.2 | |||
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 7.2 | |||
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 7.2 | |||
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 7.2 | |||
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 7.2 | |||
|- style="font-size:12pt;font-weight:bold" | |||
|style="background-color:#C5D9F1" height="15" valign="bottom" | Final uL Added | |||
| align="center" align="center" valign="bottom" | 0.96 | |||
| align="center" align="center" valign="bottom" | 0.96 | |||
| align="center" align="center" valign="bottom" | 0.48 | |||
| align="center" align="center" valign="bottom" | 0.384 | |||
| align="center" align="center" valign="bottom" | 0.192 | |||
| align="center" align="center" valign="bottom" | 0.096 | |||
| align="center" align="center" valign="bottom" | 0.048 | |||
| align="center" align="center" valign="bottom" | 0.0192 | |||
| align="center" align="center" valign="bottom" | 0.0096 | |||
|} | |||
#Run for 30 minutes at 200 V | |||
==USER/DpnII Digestion== | ==USER/DpnII Digestion== |
Revision as of 23:31, 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
Gel Quantification Check
Based on the results above (no sample after Lambda digestion), I'm going to do a massive gel dilution series to see approximately what the concentration of PPRL-A is. I'll either repeat for B, or more likely assume it is on the same scale.
Lane | 2 (Ladder) | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
Dilution | 1 | 1 | 1 | 10 | 10 | 10 | 10 | 50 | 50 |
uL Sample | 1 | 1 | 0.5 | 4 | 2 | 1 | 0.5 | 1 | 0.5 |
uL TBE | 4.5 | 4.5 | 5 | 1.5 | 3.5 | 4.5 | 5 | 4.5 | 5 |
uL buffer | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 |
Total | 7.5 | 7.5 | 7.5 | 7.5 | 7.5 | 7.5 | 7.5 | 7.5 | 7.5 |
uL Added | 7.2 | 7.2 | 7.2 | 7.2 | 7.2 | 7.2 | 7.2 | 7.2 | 7.2 |
Final uL Added | 0.96 | 0.96 | 0.48 | 0.384 | 0.192 | 0.096 | 0.048 | 0.0192 | 0.0096 |
- Run for 30 minutes at 200 V
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