Daniel:Notebook/HiResChrPaint/2013-4-27
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Probe Prep Test (Started 4/22/2013)[edit]
Ethanol Precipitation (Continued from 4/26/2013)[edit]
5. Took samples from -80 and centrifuged at 14000 rpm for 25 minutes at 4 C 6. Removed supernatant and added 750 uL 75% EtOH 7. Centrifugd at 14000 rpm for 15 minutes at 4 C 8. Removed most supernatant with pipette and then dried in hood 9. Resuspended in 8 uL nuclease free water
TBU gel Quantification[edit]
1. Preheated TBE buffer in 60 C incubator 2. microwaved for 1 minute 3. Added hot TBE to gel box and immediately preran gel for 20 minutes at 250V 4. Created samples according to following table
Lane | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
Sample | Ladder | Ladder | V4S3 | V4S3 | V6S3 | V6S3 | Ladder | Buffer |
uL sample | 0.5 | 1 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0 |
uL hot TBE | 5.5 | 5 | 5.5 | 5.5 | 5.5 | 5.5 | 5.5 | 6 |
uL 2x buffer | 6 | 6 | 6 | 6 | 6 | 6 | 6 | 6 |
loaded | 12 | 12 | 12 | 12 | 12 | 12 | 12 | 12 |
5. Incubated samples at 65C for 8 minutes 6. Cold snapped with ice box 7. Loaded 12 uL each sample and ran gel for 20 minutes at 250V 8. Stained with 3 uL SYBR gold for 25 minutes
Gel Results[edit]
Volume Report: File:GelQuant 042713.xls
File:GelQuant analysis 042713.jpg
Sample Concentrations[edit]
Sample | ng/uL | ng (7 uL) |
V4S3 | 4.6 | 32.4 |
V4S3 | 3.1 | 22.0 |
V6S3 | 3.5 | 24.7 |
Discussion[edit]
With the abysmally low yields from the Lambda Exonuclease reaction, I have come to the conclusions that it is this reaction causing the most problems in my probe prep protocol. I'm going to need to approach this reaction better before I continue. The following table summarizes the yields in (ug and %) comparing my probe preps and Noi's:
qPCR (ug) | qPCR Yield (%) | DpnII (ug) | DpnII Yield (%) | Lambda Exo (ug) | Lambda Exo Yield (%) | Yield from DPNII | |
Me | |||||||
V4S3 | 8.2 | 100 | 5.6 | 68.3 | 0.03 | 0.4 | 0.6 |
V6S3 | 7.4 | 100 | 4.7 | 63.5 | 0.02 | 0.3 | 0.5 |
Noi | |||||||
V4S3 | 13.2 | 100 | 8.9 | 67.2 | 2.2 | 16.4 | 24.4 |
V6S3 | 15.9 | 100 | 12.5 | 78.4 | 2.6 | 16.1 | 20.5 |