Daniel:Notebook/HiResChrPaint/2013-6-5
Jump to navigation
Jump to search
Probe Prep 3 (Started 5/29/2013)[edit]
DpnII Digestion[edit]
Finishing the digest on the PCR amplified probes, aa-dUTP and no aa-dUTP. Will run for 2 hours since I didn't see any qualitative difference between the two.
1. Set up
C group
Sample | uL sample | uL Dpn II (50 U/uL) | uL 10x buffer | uL H20 | total |
alpha 4-C | 35 | 2 | 5 | 8 | 50 |
alpha 6-C | 35 | 2 | 5 | 8 | 50 |
beta 4-C | 35 | 2 | 5 | 8 | 50 |
beta 6-C | 35 | 2 | 5 | 8 | 50 |
D Group
Sample | uL sample | uL Dpn II (50 U/uL) | uL 10x buffer | uL H20 | total |
alpha 4-D | 35 | 2 | 5 | 8 | 50 |
alpha 6-D | 35 | 2 | 5 | 8 | 50 |
beta 4-D | 35 | 2 | 5 | 8 | 50 |
beta 6-D | 35 | 2 | 5 | 8 | 50 |
2. Incubate in thermocycler at 37C for 16 hours; heat shock 20 minutes at 65 C 3. Column purification, elute with 30 uL EB 4. Measure in Nanodrop
Nanodrop Results[edit]
Sample | a-4C | a-6C | b-4C | b-6C | a-4D | a-6D | b-4D | b-6D |
ng/uL | 119.1 | 119.2 | 108.5 | 140.4 | 121.8 | 147.7 | 112.2 | 128.7 |
ug in 30 uL | 3.6 | 3.6 | 3.3 | 4.2 | 3.7 | 4.4 | 3.4 | 3.9 |
Yield (%) | 78.0 | 73.7 | 80.1 | 85.2 | 80.1 | 81.6 | 77.0 | 77.1 |
TBE Gel[edit]
Check to make sure samples are at least mostly digested.
1. Setup
Lane | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
Sample | 25 bp ladder | V4S2C | V6S2C | V4S2C* | V6S2C* | Undigested | V4S2D | V6S2D | V4S2D* | V6S2D* |
Note: *denotes samples without aa-dUTP
2. Ran gel for 25 minutes at 220 V 3. Stained for 1 minute with 3 uL SYBR gold