Daniel:Notebook/HiResChrPaint/2014-1-9: Difference between revisions

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===Nanodrop Results===
===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
{{GelLanes|Lane3=10bp ladder|Lane5=Alu2-B Pre-Digest|Lane7=Alu2-B Post-Digest|Lane9=Alu2-AB Post-Digest}}
{{GelLanes|Lane3=0.5 uL sample, 5.5 TBE|Lane5=1 uL sample, 5 TBE|Lane7=1 uL sample, 5 TBE|Lane9=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'''
[[Image:2014-01-09-Alu2.jpg|600px]]
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.

Latest revision as of 23:47, 9 January 2014

Alu Control (Started 1/08/2014)[edit]

Back to Calendar

DpnII Digestion[edit]

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[edit]

ALU2-AB: 37.3 ng/uL; 1.1 ug total

TBE Gel[edit]

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

File:2014-01-09-Alu2.jpg

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.