Daniel:Notebook/HiResChrPaint/2014-2-21

From ZhangLabWiki
Jump to navigation Jump to search

Multi-Color FISH (Started 02/08/2014)[edit]

Back to Calendar

qPCR of BSPP[edit]

Sample:

  • BSPP 1-DMR 220K Exp 1 (10nM); 2/8/11 from Noi
  1. Reaction table
  10 nM OligoPool 2X Kapa SYBR qPCR MM 100 uM AP1V61U 100 uM AP2V6 2 mM aa-dUTP H20 Total Volume
per rxn 0.4 50 0.4 0.4 2.5 46.3 100
BSPP 1 (6.1x) 2.44 305 2.44 2.44 15.25 282.43 610
  1. Aliquot 100 uL into each of 6 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. Pooled 3 samples together each (300 uL each, 2 total)
  4. Column Purification, elute with 40 uL EB
  5. Measure concentrations in nanodrop

qPCR Results[edit]

qPCR Curve

File:2014-02-21-BSPP1.png

Nanodrop Results

Sample ng/uL ug in 40 uL
BSPP1-A 73.9 3.0
BSPP1-B 68.4 2.7

Dye Coupling[edit]

  1. Dry samples in vacuum centrifuge
  2. Add 100 uL DMSO to vial containing ULS dye (stable for 6 months at 4C)
  *For AlexaFluor 488 label using 5 uL DMSO (stable for 1 month at 4C)
 ***VORTEX THESE SOLUTIONS VIGOROUSLY BEFORE USE EVERY TIME!
  1. Resuspend DNA in 21.5 uL labeling buffer (Component C)
    1. Measure DNA concentration in nanodrop
  2. Denature DNA at 95C for 5 minutes and snap cool on ice; centrifuge briefly
  3. Add appropriate volume of dye to sample (see table 2); if necessary add labeling buffer to bring volume to 25 uL
  4. Incubate at 80C for 15 minutes; stop the reaction by plunging the tube into an ice bath
  5. Purify using a Centri Sep column

Label Matrix

Sample Fluorophore ng/uL ug in 20 uL uL dye
V6S3 A-546 108.4 2.2 5
Cot1-High A-546 74 1.5 5
Cot1-Low A-488 36.5 0.7 1
BSPP1-A A-546 41.4 0.8 5

Labeling Results[edit]

Sample Avg Mol Weight ng/uL DNA pmol/uL dye pmol/uL DNA dye/probe base:dye ratio
V6S3 81 44.5 1.6 1.7 1.0 84
BSPP1 100 67 1.5 2.0 0.7 135
Cot1-High 800 42.4 3 0.2 18.7 43
Cot1-Low 100 28.9 0.5 0.9 0.6 175