Daniel:Notebook/HiResChrPaint/2013-10-7

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V6S3 and V4S1 Amplification Amino Primers (Started 10/03/2013)[edit]

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Dye Coupling[edit]

  • V4S1-> Alexa 546
  • V6S3-> Alexa 594

Protocol[edit]

For each, from ARES Protocol

0. Dry each sample in vacuum centrifuge, resuspend in 5 uL nfH20
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 2 uL solvent; vortex ≥10 seconds
   *dye cannot be saved for later use.  Use immediately!
5. Add 2 uL dye to sample
6. Incubate in the dark for 1 hour
7. Add 15 uL nfH20 to sample
8. Centri Sep column purification

Labeling Results[edit]

  ng/uL DNA pmol/uL dye ug DNA in 25 uL pmol/uL DNA dye/probe dye/100 bp Base:Dye Ratio (:1)
V4S1 86.1 4.3 2.2 3.2 1.3 1.6 61
V6S3 86.8 5.0 2.2 3.2 1.5 1.9 53

It would appear these numbers are slightly spurious, since it is not supposed to be possible for there to be more than one dye per probe. Likely, this means there is some leakage (although previous results would suggest that minimal free dye makes it through the separation column. Still, the labeling results are good and I have ~25 uL of 3 pmol/uL dye (which is good for 5-10 labelings according to Beliveau et al).