Daniel:Notebook/HiResChrPaint/2013-11-21

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Revision as of 16:51, 22 November 2013 by >Djacobse (→‎Positive Control 2 Started 11/14/2013))
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Positive Control 2 (Started 11/14/2013)[edit]

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ssNanodrop Results[edit]

Following is the single stranded nanodrop results from my 4 probe preps so far.

Sample ng/uL ug in 20 uL
V6S1 103.7 2.1
V6S2 226.9 4.5
V6S3 144.3 2.9
V6S4 137.2 2.7

Dye Coupling[edit]

This will be the same as usual dye couplings, except that I'm going to label all dyes with Alexa 488, and I'm going to pool two of the samples together (V4S1,V4S2 and V4S3,V4S4) together before dye coupling, since the labeling is supposed to be for 1-5 ug of DNA, and each of these only has around 2 ug.

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 141.5 6.6 3.5 5.3 1.2 1.5 65
V6S3 172 8.6 4.3 6.4 1.3 1.7 61

So everything looks great. Incindentally, I realized that if you want to do a quick calculation through from the nanodrop results (ng/uL DNA and pmol/uL dye), the optimal ratio is close to 20, which will give approximately 60 bp/dye.