Daniel:Notebook/HiResChrPaint/2013-8-2
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S1 Probe Preparation[edit]
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