Daniel:Notebook/HiResChrPaint/2013-7-18

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Revision as of 00:39, 19 July 2013 by >Djacobse
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S4 Probe Preparation

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Going off the positive results of S3 production, I'm going to expand my pool of dye-labeled probes. Since I haven't done the S4 set in a while, I'll be doing that.

PCR Amplification

1. Reaction Table
  20 nM OligoPool 2X Kapa SYBR qPCR MM 100 uM AP1V41U 100 uM AP2V4 100 uM AP1V61U 100 uM AP2V6 2 mM aa-dUTP H20 Total Volume
per rxn 0.2 50 0.4 0.4 0.4 0.4 2.5 45.7 100
V4S3 (12.5x) 2.5 625 5 5 0 0 31 581.5 1250
V6S3 (12.5x) 2.5 625 0 0 5 5 31 581.5 1250
2. Aliquot 100 uL into each of 12 qPCR tubes 
3. PCR protocol:
    i. PCR 3 min 95C
    ii. 95C 3 sec
    iii. 55C 30 sec 
    iv. 72C 20 sec
    v. plate read
    vi. goto ii x17
    vii. 72C 2 min
    viii. 16C hold
4. Pooled 3 samples together each (300 uL each, 4 pools per set, 8 total)
5. Ethanol Precipitation, added 900 uL 100% EtOH, 30 uL 3 M NaOAc, 1 uL glycoblue
6. Incubated 4 hours; resuspend in 50 uL nfH20
7. Column Purification, elute with 40 uL EB
8. Measure concentrations in nanodrop

qPCR Results

File:2013-7-18-S4.png

Nanodrop Results

Sample V4S4A V4S4B V4S4C V4S4D V6S4A V6S4B V6S4C V6S4D
ng/uL 87.9 89.4 83.9 86.9 101.8 102.7 89.2 89.1
ug in 40 uL 3.5 3.6 3.4 3.5 4.1 4.1 3.6 3.6
Total ug 13.9 15.3

DpnII Digestion

1. Prepare Samples
Sample V4S4A V4S4B V4S4C V4S4D V6S4A V6S4B V6S4C V6S4D
ng/uL 87.9 89.4 83.9 86.9 101.8 102.7 89.2 89.1
ug in 35 uL 3.1 3.1 2.9 3.0 3.6 3.6 3.1 3.1
uL 10X buffer 5 5 5 5 5 5 5 5
uL DpnII (50U/uL) 2 2 2 2 2 2 2 2
uL nfH2O 8 8 8 8 8 8 8 8
Total 50 50 50 50 50 50 50 50
2. Incubate samples at 37C for 16 hours; 20 minute heat shock at 65C; Continued 7/19/2013]]