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...") |
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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''' | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#FCD5B4;font-size:12pt;font-weight:bold" align="center" | |||
| width="65" height="30" | Component | |||
| width="65" | 1X Reaction | |||
| width="65" | 24.5X Scaled | |||
|- style="font-size:12pt" | |||
|style="background-color:#C5D9F1" height="30" valign="bottom" | Padlock Probes | |||
| align="center" align="center" valign="bottom" | 0.2 | |||
| align="center" align="center" valign="bottom" | 4.9 | |||
|- style="font-size:12pt" | |||
|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" | 9.8 | |||
|- style="font-size:12pt" | |||
|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" | 9.8 | |||
|- style="font-size:12pt" | |||
|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" | 1225 | |||
|- style="font-size:12pt" | |||
|style="background-color:#C5D9F1" height="15" valign="bottom" | H2O | |||
| align="center" align="center" valign="bottom" | 49 | |||
| align="center" align="center" valign="bottom" | 1200.5 | |||
|- style="font-size:12pt;font-weight:bold" | |||
|style="background-color:#C5D9F1" height="15" valign="bottom" | Total | |||
| align="center" align="center" valign="bottom" | 100 | |||
| align="center" align="center" valign="bottom" | 2450 | |||
|} | |||
#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 | |||
==USER/DpnII Digestion== | ==USER/DpnII Digestion== |
Revision as of 15:24, 18 August 2014
Padlock Probes (Started 8/13/2014)
qPCR
I need more padlock probes, so I'm going to repeat qPCR with 24x samples.
Reaction Table
Component | 1X Reaction | 24.5X Scaled |
Padlock Probes | 0.2 | 4.9 |
100 uM pAP1V41U | 0.4 | 9.8 |
100 uM pAP2V4 | 0.4 | 9.8 |
KAPA SYBR Fast Master Mix (2X) | 50 | 1225 |
H2O | 49 | 1200.5 |
Total | 100 | 2450 |
- 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
USER/DpnII Digestion
Reaction Table
Reagent | uL |
amplicon | 35 |
USER | 5 |
nf H2O | 40 |
Total | 80 |
- Set up reaction according to table in a 0.2 mL PCR tube
- Incubate for 2 hours at 37C
- Add 15 uL according to DpnII Primer table (below)
DpnII Primer Table
Reagent | uL |
10X DpnII Buffer | 10 |
100 uM RE-DpnII-V4 guide oligo | 5 |
Total | 15 |
- Incubate at 94C for 2 minutes
- Cool to 37C and incubate for 3 minutes
- Add 5 uL DpnII and incubate at 37C for 2 hours
- Heat inactivate DpnII at 65C for 20 minutes