Daniel:Notebook/HiResChrPaint/2014-2-21: Difference between revisions

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>Djacobse
>Djacobse
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| align="center" | 0.8
| align="center" | 0.8
| align="center" | 5
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===Labeling Results===
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="font-size:12pt;font-weight:bold" align="center"
| width="65" height="30" | Sample
| width="65" | Avg Mol Weight
| width="65" | ng/uL DNA
| width="65" | pmol/uL dye
| width="65" | pmol/uL DNA
| width="65" | dye/probe
| width="65" | base:dye ratio
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="15" | V6S3
| align="center" | 81
| align="center" | 44.5
| align="center" | 1.6
| align="center" | 1.7
| align="center" | 1.0
| align="center" | 84
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="15" | BSPP1
| align="center" | 100
| align="center" | 67
| align="center" | 1.5
| align="center" | 2.0
| align="center" | 0.7
| align="center" | 135
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="15" | Cot1-High
| align="center" | 800
| align="center" | 42.4
| align="center" | 3
| align="center" | 0.2
| align="center" | 18.7
| align="center" | 43
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="15" | Cot1-Low
| align="center" | 100
| align="center" | 28.9
| align="center" | 0.5
| align="center" | 0.9
| align="center" | 0.6
| align="center" | 175


|}
|}

Revision as of 00:23, 23 February 2014

Multi-Color FISH (Started 02/08/2014)

Back to Calendar

qPCR of BSPP

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

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

  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

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