Daniel:Notebook/HiResChrPaint/2013-7-16: Difference between revisions
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2. Incubate samples at 37C for 16 hours; 20 minute heat shock at 65C; [[Daniel:Notebook/HiResChrPaint/2013- | 2. Incubate samples at 37C for 16 hours; 20 minute heat shock at 65C; [[Daniel:Notebook/HiResChrPaint/2013-7-17|Continued]] 7/17/2013]] |
Latest revision as of 18:35, 17 July 2013
S3 Probe Preparation[edit]
Since I don't have any S3 set left, I'm going to make a new run using the various new techniques, specifically I am more confident in the dye coupling stage now. I'm going to make just the aa-dUTP (ARES labeling) style for now, since it is difficult to balance the reactions when I have a lot of samples.
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 |
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 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[edit]
Nanodrop Results[edit]
Sample | V4S1A | V4S1B | V4S1C | V4S1D | V6S1A | V6S1B | V6S1C | V6S1D |
ng/uL | 97.6 | 91.6 | 99 | 99.8 | 118.6 | 113.8 | 117.7 | 118.8 |
ug in 40 uL | 3.9 | 3.7 | 4.0 | 4.0 | 4.7 | 4.6 | 4.7 | 4.8 |
Total ug | 15.5 | 18.8 |
DpnII Digestion[edit]
1. Prepare Samples
Sample | V4S3A | V4S3B | V4S3C | V4S3D | V6S3A | V6S3B | V6S3C | V6S3D |
ng/uL | 97.6 | 91.6 | 99 | 99.8 | 118.6 | 113.8 | 117.7 | 118.8 |
ug in 35 uL | 3.4 | 3.2 | 3.5 | 3.5 | 4.2 | 4.0 | 4.1 | 4.2 |
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 7/17/2013]]