Daniel:Notebook/HiResChrPaint/2013-8-2

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S1 Probe Preparation[edit]

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Armed with a new bottle of water (seriously...), I'm now ready to amplify the S1 set.

PCR Amplification[edit]

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
V4S1 (12.5x) 2.5 625 5 5 0 0 31 581.5 1250
V6S1 (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. Pool 3 samples together each (300 uL each, 4 pools per set, 8 total)
5. Column Purification, elute with 40 uL EB
6. Measure concentrations in nanodrop

qPCR Results[edit]

Nanodrop Results[edit]

Sample V4S1A V4S1B V4S1C V4S1D V6S1A V6S1B V6S1C V6S1D
ng/uL 96.8 92.3 90.1 97.2 141.6 148.1 142 136.3
ug in 40 uL 3.9 3.7 3.6 3.9 5.7 5.9 5.7 5.5
Total ug       15.1       22.7

DpnII Digestion[edit]

1. Prepare Samples
Sample V4S1A V4S1B V4S1C V4S1D V6S1A V6S1B V6S1C V6S1D
ng/uL 96.8 92.3 90.1 97.2 141.6 148.1 142 136.3
ug in 35 uL 3.4 3.2 3.2 3.4 5.0 5.2 5.0 4.8
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 2 hours; 20 minute heat shock at 65C
3. Column purification, elute with 30 uL EB
4. Measure in Nanodrop

Nanodrop Results[edit]

Sample V4S1A V4S1B V4S1C V4S1D V6S1A V6S1B V6S1C V6S1D
ng/uL 88.2 87.1 77.4 94.7 129.9 142.6 139.6 132.3
ug in 30 uL 2.6 2.6 2.3 2.8 3.9 4.3 4.2 4.0
Total ug       10.4       16.3

TBE Gel[edit]

Lane 1 2 3 4 5 6 7 8 9 10 11 12
Sample 10 bp ladder V4S1A V4S1B V4S1C V4S1D V6S1A V6S1B V6S1C V6S1D

Gel Image