Daniel:Notebook/HiResChrPaint/2014-2-21: Difference between revisions
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'''qPCR Curve''' | '''qPCR Curve''' | ||
[[Image:| | [[Image:2014-02-21-BSPP1.png|500px]] | ||
'''Nanodrop Results''' | '''Nanodrop 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="15" | Sample | |||
| width="65" | ng/uL | |||
| width="65" | ug in 40 uL | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | BSPP1-A | |||
| align="center" | 73.9 | |||
| align="center" | 3.0 | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | BSPP1-B | |||
| align="center" | 68.4 | |||
| align="center" | 2.7 | |||
|} | |||
==Dye Coupling== | |||
#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 [[Daniel:Protocols/AlexaDyeCouple#ULYSIS|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 [[Daniel:Protocols/CentriSep|Centri Sep column]] | |||
'''Label Matrix''' | |||
{| 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="15" | Sample | |||
| width="73" | Fluorophore | |||
| width="65" | ng/uL | |||
| width="65" | ug in 20 uL | |||
| width="65" | uL dye | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | V6S3 | |||
| A-546 | |||
| align="center" | 108.4 | |||
| align="center" | 2.2 | |||
| align="center" | 5 | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | Cot1-High | |||
| A-546 | |||
| align="center" | 74 | |||
| align="center" | 1.5 | |||
| align="center" | 5 | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | Cot1-Low | |||
| A-488 | |||
| align="center" | 36.5 | |||
| align="center" | 0.7 | |||
| align="center" | 1 | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | BSPP1-A | |||
| A-546 | |||
| align="center" | 41.4 | |||
| align="center" | 0.8 | |||
| align="center" | 5 | |||
|} | |||
===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 | |||
|} | |||
[[Category:HRCP]] [[Category:20140208]] [[Category:qPCR]] [[Category:ULYSIS Label]] |
Latest revision as of 18:24, 2 July 2014
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 |