Daniel:Notebook/HiResChrPaint/2014-1-9
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Alu Control (Started 1/08/2014)
DpnII Digestion
I'm going to do a test digestion/gel to make sure this works. Since I had such low input I'm going to use 2 samples and pool after the digestion.
1. Reaction Table
Sample | ALU2-A | ALU2-B |
ng/uL | 25.1 | 25 |
ug in 35 uL | 0.9 | 0.9 |
uL 10X buffer | 5 | 5 |
uL DpnII (50U/uL) | 1 | 1 |
uL nfH2O | 9 | 9 |
Total | 50 | 50 |
2. Incubate samples at 37C for 2 hours; 20 minute heat shock at 65C 3. Column Purification, elute with 30 uL EB 4. Measure concentrations in nanodrop
Nanodrop Results
ALU2-AB: 37.3 ng/uL; 1.1 ug total
TBE Gel
1. For Each Sample: 2 uL dye; load 7.2 uL
Lane | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
Sample | 10bp ladder | Alu2-B Pre-Digest | Alu2-B Post-Digest | Alu2-AB Post-Digest |
Lane | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
Sample | 0.5 uL sample, 5.5 TBE | 1 uL sample, 5 TBE | 1 uL sample, 5 TBE | 0.5 uL sample, 5.5 TBE |
2. Run gel for 27 minutes at 220V 3. Stain 2 minutes with 3 uL SYBR gold
Gel Image
This gel image implies that the Alu DNA is not being cut with DpnII. From a look at my primer sequences, there is actually no cut site (it must have been ordered into each of the probe sequences. This will produce a non-matching overlap at the 3' end of each probe, but other than that should yield no real problems. Another point to test should be lambda exonuclease digestion, but we have not, as of yet, determined a good way to tell whether or not exonuclease digestion is actually generating single stranded probes.