Daniel:Notebook/HiResChrPaint/2013-9-26
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Amino Group Primers
So the new primers are in. We ordered them a few weeks ago. All the primers have a 5' amino group, which should be compatible with the Alexa-dye coupling system. Thanks to Matt's results yesterday, I know that when the dye coupling does not work, the Centri-Sep columns yield very little dye. So, before going nuts on making new probes, I'm going to test the water with a simple comparison. For sample 1, I'm going to test the dye using the new primer, plus no aa-dUTP. I'm also going to do a positive control using a standard amplification using the old primers and aa-dUTP. Finally, I'll run a negative control using old primer and no aa-dUTP. I'll do this for both V4S3 and V6S3, since I should test both primer sets.
Amino Primers Test
qPCR
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 | |
std 100 uL rxn | 0.2 | 50 | 0.4 | 0.4 | 0.4 | 0.4 | 2.5 | 46 | 100 |
V4S3* (2.5x) | 0.5 | 125 | 1 | 1 | 0 | 0 | 0 | 122.5 | 250 |
V6S3* (2.5x) | 0.5 | 125 | 0 | 0 | 1 | 1 | 0 | 122.5 | 250 |
V6S3 (2.5x) | 0.5 | 125 | 0 | 0 | 1 | 1 | 6.25 | 116.25 | 250 |
V6S3- (2.5x) | 0.5 | 125 | 0 | 0 | 1 | 1 | 0 | 122.5 | 250 |
2. Aliquot 100 uL into each of 2 qPCR tubes (8 in total) 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. Pool 2 samples together (200 uL each) 5. Column Purification, elute with 40 uL EB 6. Measure in nanodrop
Dye Coupling
- Following ARES Protocol
0. Dry samples in vacuum centrifuge 1.