Daniel:Notebook/HiResChrPaint/2014-8-19
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Padlock Probes (Started 8/13/2014)[edit]
Ethanol Precipitation (From 8/18/2014)[edit]
- 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[edit]
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[edit]
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
- Heat inactivate for 10 minutes at 75C
- Purify using 2 ssDNA columns, elute with 20 uL nfH2O each
Nanodrop Results[edit]
Sample A reported 2.1 ng/uL and sample B reported 1.7 ng/uL. This means just about everything was lost.
Gel Quantification Check[edit]
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
Gel Image