Daniel:Notebook/HiResChrPaint/2013-7-22
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S2 Probe Preparation
Despite mixed reviews from the S4 set, I'm going to go ahead and make the S2 set. Also, I talked with Alan and he and I are going to prep some cells tomorrow. I'll probably go ahead and attempt fish with the S3 set, since that one worked out great. If I have other probes available, I may use them as well, but Alan and I won't be preparing the cells until tomorrow afternoon, which gives me plenty of time to prep a new set.
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
Nanodrop Results
Sample | V4S4A | V4S4B | V4S4C | V4S4D | V6S4A | V6S4B | V6S4C | V6S4D |
ng/uL | 52.6 | 44.5 | 52.1 | 47.4 | 105.7 | 111.3 | 98.1 | 103.3 |
ug in 40 uL | 2.1 | 1.8 | 2.1 | 1.9 | 4.2 | 4.5 | 3.9 | 4.1 |
Total ug | 5.8 | 12.5 |
DpnII Digestion
1. Prepare Samples
2. Incubate samples at 37C for 16 hours; 20 minute heat shock at 65C; Continued 7/23/2013]]