Daniel:Notebook/HiResChrPaint/2014-2-21
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Multi-Color FISH (Started 02/08/2014)[edit]
qPCR of BSPP[edit]
Sample:
- BSPP 1-DMR 220K Exp 1 (10nM); 2/8/11 from Noi
- 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 |
- Aliquot 100 uL into each of 6 qPCR tubes
- PCR protocol:
- PCR 3 min 95C
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto ii x17
- 72C 2 min
- 16C hold
- Pooled 3 samples together each (300 uL each, 2 total)
- Column Purification, elute with 40 uL EB
- Measure concentrations in nanodrop
qPCR Results[edit]
qPCR Curve
Nanodrop Results
Sample | ng/uL | ug in 40 uL |
BSPP1-A | 73.9 | 3.0 |
BSPP1-B | 68.4 | 2.7 |
Dye Coupling[edit]
- Dry samples in vacuum centrifuge
- 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!
- Resuspend DNA in 21.5 uL labeling buffer (Component C)
- Measure DNA concentration in nanodrop
- Denature DNA at 95C for 5 minutes and snap cool on ice; centrifuge briefly
- Add appropriate volume of dye to sample (see table 2); if necessary add labeling buffer to bring volume to 25 uL
- Incubate at 80C for 15 minutes; stop the reaction by plunging the tube into an ice bath
- 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 |