Daniel:Notebook/HiResChrPaint/2014-8-18: Difference between revisions
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| width="65" height="30" | Component | | width="65" height="30" | Component | ||
| width="65" | 1X Reaction | | width="65" | 1X Reaction | ||
| width="65" | | | width="65" | 12.2X Scaled | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
|style="background-color:#C5D9F1" height="30" valign="bottom" | Padlock Probes | |style="background-color:#C5D9F1" height="30" valign="bottom" | Padlock Probes | ||
| align="center" align="center" valign="bottom" | 0.2 | | align="center" align="center" valign="bottom" | 0.2 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 2.44 | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
|style="background-color:#C5D9F1" height="30" valign="bottom" | 100 uM pAP1V41U | |style="background-color:#C5D9F1" height="30" valign="bottom" | 100 uM pAP1V41U | ||
| align="center" align="center" valign="bottom" | 0.4 | | align="center" align="center" valign="bottom" | 0.4 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 4.88 | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
|style="background-color:#C5D9F1" height="30" valign="bottom" | 100 uM pAP2V4 | |style="background-color:#C5D9F1" height="30" valign="bottom" | 100 uM pAP2V4 | ||
| align="center" align="center" valign="bottom" | 0.4 | | align="center" align="center" valign="bottom" | 0.4 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 4.88 | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
|style="background-color:#C5D9F1" height="45" valign="bottom" | KAPA SYBR Fast Master Mix (2X) | |style="background-color:#C5D9F1" height="45" valign="bottom" | KAPA SYBR Fast Master Mix (2X) | ||
| align="center" align="center" valign="bottom" | 50 | | align="center" align="center" valign="bottom" | 50 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 610 | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
|style="background-color:#C5D9F1" height="15" valign="bottom" | H2O | |style="background-color:#C5D9F1" height="15" valign="bottom" | H2O | ||
| align="center" align="center" valign="bottom" | 49 | | align="center" align="center" valign="bottom" | 49 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 597.8 | ||
|- style="font-size:12pt;font-weight:bold" | |- style="font-size:12pt;font-weight:bold" | ||
|style="background-color:#C5D9F1" height="15" valign="bottom" | Total | |style="background-color:#C5D9F1" height="15" valign="bottom" | Total | ||
| align="center" align="center" valign="bottom" | 100 | | align="center" align="center" valign="bottom" | 100 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 1220 | ||
|} | |} | ||
Line 57: | Line 57: | ||
##72C 2 min | ##72C 2 min | ||
##Hold at 16C forever | ##Hold at 16C forever | ||
#Ethanol Precipitation | #[[Daniel:Protocols/EtOHPrecip|Ethanol Precipitation]] | ||
##Continued [[Daniel:Notebook/HiResChrPaint/2014-8-19|tomorrow]] | |||
== | ===qPCR Results=== | ||
[[Image:2014-08-18_PPRL.png|Successful run on all lanes|500px]] | |||
| | |||
[[Category:HRCP]] [[Category:qPCR]] [[Category:20140813]] | |||
Latest revision as of 22:38, 9 September 2014
Padlock Probes (Started 8/13/2014)[edit]
qPCR[edit]
I need more padlock probes, so I'm going to repeat qPCR with 24x samples.
Reaction Table
Component | 1X Reaction | 12.2X Scaled |
Padlock Probes | 0.2 | 2.44 |
100 uM pAP1V41U | 0.4 | 4.88 |
100 uM pAP2V4 | 0.4 | 4.88 |
KAPA SYBR Fast Master Mix (2X) | 50 | 610 |
H2O | 49 | 597.8 |
Total | 100 | 1220 |
- Prepare master mix according to table above
- Add 100 uL reaction to PCR tubes (5 total)
- qPCR Protocol
- 95C 30 sec
- 95C 10 sec
- 55C 20 sec
- 72C 30 sec
- goto (3.2) 17 times
- 72C 2 min
- Hold at 16C forever
- Ethanol Precipitation
- Continued tomorrow