Daniel:Notebook/HiResChrPaint/2013-9-26

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Amino Group Primers[edit]

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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[edit]

qPCR[edit]

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[edit]

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[edit]

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.