Daniel:Notebook/HiResChrPaint/2013-6-5

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Probe Prep 3 (Started 5/29/2013)[edit]

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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

File:DpnDigest 060513.jpg