Daniel:Notebook/HiResChrPaint/2013-7-16: Difference between revisions
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4. Pooled 3 samples together each (300 uL each, 4 pools per set, 8 total) | 4. Pooled 3 samples together each (300 uL each, 4 pools per set, 8 total) | ||
5. [[Daniel:Protocols/EtOHPrecip|Ethanol Precipitation]], added 900 uL 100% EtOH, 30 uL 3 M NaOAc, 1 uL glycoblue | 5. [[Daniel:Protocols/EtOHPrecip|Ethanol Precipitation]], added 900 uL 100% EtOH, 30 uL 3 M NaOAc, 1 uL glycoblue | ||
6. Incubated | 6. Incubated 4 hours; resuspend in 50 uL nfH20 | ||
7. [[Daniel:Protocols/Qiaquick_Column|Column Purification]], elute with 40 uL EB | 7. [[Daniel:Protocols/Qiaquick_Column|Column Purification]], elute with 40 uL EB | ||
8. Measure concentrations in nanodrop | 8. Measure concentrations in nanodrop |
Revision as of 22:52, 16 July 2013
S3 Probe Preparation
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
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