Daniel:Notebook/HiResChrPaint/2013-6-20: Difference between revisions
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| align="center" | 4.896 | | align="center" | 4.896* | ||
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| align="center" | 14.38 | | align="center" | 14.38** | ||
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Notes: *1.7x yield (in ug) of qPCR of V4S1 using 800 uL starting material; ** 2.1x yield (in ug) of qPCR of V6S1 using 800 uL starting material |
Revision as of 00:11, 21 June 2013
S1 Probe Preparation
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 |
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 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 2 hours; resuspend in 50 uL nfH20 7. Column Purification, elute with 40 uL EB 8. Measure concentrations in nanodrop
qPCR Results
Nanodrop Results
Sample | V4S1A | V4S1B | V4S1C | V4S1D | V6S1A | V6S1B | V6S1C | V6S1D |
ng/uL | 30.7 | 31.1 | 31.5 | 29.1 | 90 | 87.7 | 91.9 | 89.9 |
ug in 40 uL | 1.228 | 1.244 | 1.26 | 1.164 | 3.6 | 3.508 | 3.676 | 3.596 |
Total ug | 4.896* | 14.38** |
Notes: *1.7x yield (in ug) of qPCR of V4S1 using 800 uL starting material; ** 2.1x yield (in ug) of qPCR of V6S1 using 800 uL starting material