Daniel:Notebook/HiResChrPaint/2013-9-26
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 | 46.5 | 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. Warm DMSO and bicarbonate labeling buffer and nuclease free water to room temperature 2. Add 5 uL DNA and 3 uL of sodium bicarbonate buffer 3. Denature samples for 5 minutes at 95C, then snap cool using ice box 4. Dissolve 1 vial of reactive dye in 8 uL solvent; vortex ≥10 seconds *dye cannot be saved for later use. Use immediately! 5. Add 2 uL dye to each sample 6. Incubate in the dark for 1 hour 7. Add 15 uL nfH20 to sample 8. Centri-Sep purification 9. Measure in nanodrop
Results
Sample | Post PCR ng/uL | Post PCR ug | Pre Spin ng/uL | Pre Spin ug | PreSpin pmol/uL ARES 546 | Post Spin ng/uL | Post Spin pmol/uL DNA | Post Spin ug | Post spin pmol/uL ARES 546 | Post Spin BP/dye | Dye/Probe |
Am V4S3 | 55.5 | 2.2 | 12.4 | 6.2 | 11.4 | 51.4 | 0.8 | 1.3 | 0.5 | 312 | 0.7 |
Am V6S3 | 63.3 | 2.5 | 10.9 | 5.5 | 9.8 | 72 | 1.1 | 1.8 | 0.6 | 364 | 0.6 |
V6S3 aa-dUTP | 57.1 | 2.3 | 12.7 | 6.4 | 11.4 | 58.4 | 0.9 | 1.5 | 0.6 | 295 | 0.7 |
V6S3 | 61.9 | 2.5 | 10 | 5.0 | 10 | 61.9 | 0.9 | 1.5 | 0.1 | 1876 | 0.1 |
So it would appear that the amino primers do work. I'll have to do a formal run, however, before I can tell if they are truly as effective as using aa-dUTP. The main concern I have right now is that several aa-dUTP can be incorporated per probe, where as only 1 dye can be incorporated per probe with the primers. Therefore, the maximum dye/probe for amino modified primers is 1, but I have frequently had >1 (though close to 1) dye/probe with the aa-dUTP.