Daniel:Notebook/HiResChrPaint/2014-7-8

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Strand Control[edit]

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Andrew has pointed out to me that I should always do the processing on the probes, just to keep results consistent. I am fairly confident this is not the cause of my difficulties, but it is still wise to keep that particular aspect consistent. As such, I will be running through the more rigorous method of probe prep.

qPCR[edit]

I'll be running through V6S2 and V6S3, my favorites.

  1. Reaction Table
  10 nM OligoPool 2X Kapa SYBR qPCR MM 100 uM AP1V61U 100 uM AP2V6 H20 Total Volume
per rxn 0.4 50 0.4 0.4 48.8 100
V6S2 (12.2x) 4.88 610 4.88 4.88 595.36 1220
V6S3 (12.2x) 4.88 610 4.88 4.88 595.36 1220
  1. Aliquot 100 uL into each of 12 qPCR tubes
  2. PCR protocol:
    1. PCR 3 min 95C
    2. 95C 3 sec
    3. 55C 30 sec
    4. 72C 20 sec
    5. plate read
    6. goto ii x17
    7. 72C 2 min
    8. 16C hold
  3. Pool 3 samples together
  4. Column Purification, elute with 40 uL EB
  5. Measure concentrations in nanodrop

qPCR Results[edit]

This is a caption.

Nanodrop Results

  V6S2A V6S2B V6S2C V6S2D V6S3A V6S3B V6S3C V6S3D
ng/uL 99.9 95.4 94.6 84.5 110 112.2 111.6 99.8
ug in 40 uL 4.0 3.8 3.8 3.4 4.4 4.5 4.5 4.0
ug total 15.0 17.3
                 

DpnII Digestion[edit]

  1. Reaction Table
  V6S2A V6S2B V6S2C V6S2D V6S3A V6S3B V6S3C V6S3D
ng/uL 99.9 95.4 94.6 84.5 110 112.2 111.6 99.8
ug in 35 uL 3.5 3.3 3.3 3.0 3.9 3.9 3.9 3.5
uL sample 35 35 35 35 35 35 35 35
uL 10X buffer 5 5 5 5 5 5 5 5
uL nfH20 8 8 8 8 8 8 8 8
uL DpnII (10 U/uL) 2 2 2 2 2 2 2 2
Total 50 50 50 50 50 50 50 50
  1. Incubate 16 hours at 37C
  2. Heat inactivate 20 minutes at 65C
  3. Continued tomorrow