Daniel:Notebook/HiResChrPaint/2014-8-18: Difference between revisions
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>Djacobse (Created page with "=Padlock Probes (Started 8/17/2014)= Back to Calendar ==USER/DpnII Digestion== '''Reaction Ta...") |
>Djacobse |
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(5 intermediate revisions by the same user not shown) | |||
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=Padlock Probes ([[Daniel:Notebook/HiResChrPaint/2014-8-13|Started 8/ | =Padlock Probes ([[Daniel:Notebook/HiResChrPaint/2014-8-13|Started 8/13/2014]])= | ||
[[ Daniel:Notebook/HiResChrPaint|Back to Calendar]] | [[ Daniel:Notebook/HiResChrPaint|Back to Calendar]] | ||
== | ==qPCR== | ||
I need more padlock probes, so I'm going to repeat qPCR with 24x samples. | |||
'''Reaction Table''' | '''Reaction Table''' | ||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | ||
|- style="background-color:# | |- style="background-color:#FCD5B4;font-size:12pt;font-weight:bold" align="center" | ||
| width=" | | width="65" height="30" | Component | ||
| width="65 | | width="65" | 1X Reaction | ||
| width="65" | 12.2X Scaled | |||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
|style="background-color:# | |style="background-color:#C5D9F1" height="30" valign="bottom" | Padlock Probes | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 0.2 | ||
| align="center" align="center" valign="bottom" | 2.44 | |||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
|style="background-color:# | |style="background-color:#C5D9F1" height="30" valign="bottom" | 100 uM pAP1V41U | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 0.4 | ||
| align="center" align="center" valign="bottom" | 4.88 | |||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
|style="background-color:# | |style="background-color:#C5D9F1" height="30" valign="bottom" | 100 uM pAP2V4 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 0.4 | ||
| align="center" align="center" valign="bottom" | 4.88 | |||
| align="center" align="center" valign="bottom" | | |||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
|style="background-color:# | |style="background-color:#C5D9F1" height="45" valign="bottom" | KAPA SYBR Fast Master Mix (2X) | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 50 | ||
| align="center" align="center" valign="bottom" | 610 | |||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
|style="background-color:# | |style="background-color:#C5D9F1" height="15" valign="bottom" | H2O | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 49 | ||
| 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:# | |style="background-color:#C5D9F1" height="15" valign="bottom" | Total | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 100 | ||
| align="center" align="center" valign="bottom" | 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 | |||
#[[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