Daniel:Notebook/HiResChrPaint/2014-8-18: Difference between revisions

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>Djacobse
>Djacobse
 
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##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===
===qPCR Results===
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[[Image:2014-08-18_PPRL.png|Successful run on all lanes|500px]]
[[Image:2014-08-18_PPRL.png|Successful run on all lanes|500px]]


==USER/DpnII Digestion==
[[Category:HRCP]] [[Category:qPCR]] [[Category:20140813]]
 
'''Reaction Table'''
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="background-color:#B8CCE4;font-size:12pt"
| width="86" height="15"  valign="bottom" | Reagent
| width="65" align="center" valign="bottom" | uL
 
|- style="font-size:12pt"
|style="background-color:#FCD5B4" height="15"  valign="bottom" | amplicon
| align="center" align="center" valign="bottom" | 35
 
|- style="font-size:12pt"
|style="background-color:#FCD5B4" height="15"  valign="bottom" | USER
| align="center" align="center" valign="bottom" | 5
 
|- style="font-size:12pt"
|style="background-color:#FCD5B4" height="15"  valign="bottom" | nf H2O
| align="center" align="center" valign="bottom" | 40
 
|- style="font-size:12pt;font-weight:bold"
|style="background-color:#FCD5B4" height="15"  valign="bottom" | Total
| align="center" align="center" valign="bottom" | 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'''
 
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="background-color:#B8CCE4;font-size:12pt"
| width="91" height="15"  valign="bottom" | Reagent
| width="65" align="center" valign="bottom" | uL
 
|- style="font-size:12pt"
|style="background-color:#FCD5B4" height="15"  valign="bottom" | 10X DpnII Buffer
| align="center" align="center" valign="bottom" | 10
 
|- style="font-size:12pt"
|style="background-color:#FCD5B4" height="30"  valign="bottom" | 100 uM RE-DpnII-V4 guide oligo
| align="center" align="center" valign="bottom" | 5
 
|- style="font-size:12pt;font-weight:bold"
|style="background-color:#FCD5B4" height="15"  valign="bottom" | Total
| align="center" align="center" valign="bottom" | 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

Latest revision as of 22:38, 9 September 2014

Padlock Probes (Started 8/13/2014)[edit]

Back to Calendar

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
  1. Prepare master mix according to table above
  2. Add 100 uL reaction to PCR tubes (5 total)
  3. qPCR Protocol
    1. 95C 30 sec
    2. 95C 10 sec
    3. 55C 20 sec
    4. 72C 30 sec
    5. goto (3.2) 17 times
    6. 72C 2 min
    7. Hold at 16C forever
  4. Ethanol Precipitation
    1. Continued tomorrow

qPCR Results[edit]

Successful run on all lanes