Daniel:Notebook/HiResChrPaint/2014-8-19

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Padlock Probes (Started 8/13/2014)[edit]

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Ethanol Precipitation (From 8/18/2014)[edit]

  1. Remove from -80C centrifuge at 4400 rpm for 30 minutes at 4C
  2. Add 700 uL 75% EtOH
    1. Break up the pellet and transfer to a 1.5mL tube
  3. Centrifuge once more at 14000 rpm for 10 minutes
  4. Take out supernatant and dry in laminar flow hood for 20 minutes
  5. 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
  1. Set up reaction according to table
  2. Incubate at 37C for 1 hour
  3. Heat inactivate for 10 minutes at 75C
  4. 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
  1. Run for 30 minutes at 200 V

Gel Image

File:2014-08-19-PPRL-A.jpg