Daniel:Notebook/HiResChrPaint/2013-6-3
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Probe Prep 3 (Started 5/29/2013)[edit]
Gel for DpnII Digestion[edit]
Looking to see if beta group (no aa-dUTP) has been digestion efficiency than alpha group (with aa-dUTP).
1. Set up 0.5 uL sample, 2 uL 6x dye, 5.5 uL TBE
Lane | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 |
Sample | 10 bp ladder | V4S2 | V6S2 | V4S2* | V6S2* | 10 bp ladder | V4S2 | V6S2 | V4S2* | V6S2* |
- denotes DNA without aa-dUTP (beta group)
2. Run for 25 minutes at 220V 3. Stain for 3 minutes with 3 uL SYBR gold
Gel Image[edit]
File:DpnDigest 060313 aadUTPvsno.jpg
Discussion[edit]
From this image it is not clear whether or not the aa-dUTP is making a difference. I'll try again with the 16 hour digestion to see if it is any better.
DpnII Digestion Test (16 hours)[edit]
Only running 1/4 of DNA I have for each sample to compare DpnII reaction yield using aadUTP DNA and normal DNA. Using longer incubation time.
1. Set up
Sample | uL sample | uL Dpn II (50 U/uL) | uL 10x buffer | uL H20 | total |
alpha 4-B | 35 | 2 | 5 | 8 | 50 |
alpha 6-B | 35 | 2 | 5 | 8 | 50 |
beta 4-B | 35 | 2 | 5 | 8 | 50 |
beta 6-B | 35 | 2 | 5 | 8 | 50 |
2. Incubate in thermocycler at 37C for 16 hours; heat shock 20 minutes at 65 C 3. Continued 6/4/2013