Daniel:Notebook/HiResChrPaint/2014-9-10: Difference between revisions
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>Djacobse (Created page with "=Padlock Probes II= Back to Calendar ===Nanodrop Results=== ==DpnII/USER Digestion==") |
>Djacobse |
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==DpnII/USER Digestion== | ==DpnII/USER Digestion== | ||
'''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;font-weight:bold" align="center" | |||
| width="93" height="33" | Reagent | |||
| width="65" | PPRL (uL) | |||
|- style="font-size:12pt" | |||
|style="background-color:#FCD5B4" height="15" valign="bottom" | amplicon | |||
| align="center" align="center" valign="bottom" | 30 | |||
|- style="font-size:12pt" | |||
|style="background-color:#FCD5B4" height="15" valign="bottom" | ug Input | |||
| align="center" align="center" valign="bottom" | 2.0 | |||
|- 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" | 45 | |||
|- 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;font-weight:bold" align="center" | |||
| width="93" height="31" | Reagent | |||
| width="65" | PPRL (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 | |||
===Nanodrop Results=== | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#CCC0DA;font-size:12pt" | |||
| width="86" height="28" valign="bottom" | Sample | |||
| width="65" align="center" | ng/uL ssDNA | |||
| width="65" align="center" | ug in 20 uL | |||
| width="65" align="center" | Total | |||
|- style="font-size:12pt" | |||
|style="background-color:#D9D9D9" height="15" valign="bottom" | PPRL-DpnII-A | |||
| align="center" align="center" valign="bottom" | 38.7 | |||
| align="center" align="center" valign="bottom" | 0.8 | |||
| align="center" align="center" valign="bottom" | | |||
|- style="font-size:12pt" | |||
|style="background-color:#D9D9D9" height="15" valign="bottom" | PPRL-DpnII-B | |||
| align="center" align="center" valign="bottom" | 35.3 | |||
| align="center" align="center" valign="bottom" | 0.7 | |||
|style="font-weight:bold" align="center" align="center" valign="bottom" | 1.5 | |||
|} |
Revision as of 17:52, 10 September 2014
Padlock Probes II
Nanodrop Results
DpnII/USER Digestion
Reaction Table
Reagent | PPRL (uL) |
amplicon | 30 |
ug Input | 2.0 |
USER | 5 |
nf H2O | 45 |
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 | PPRL (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
Nanodrop Results
Sample | ng/uL ssDNA | ug in 20 uL | Total |
PPRL-DpnII-A | 38.7 | 0.8 | |
PPRL-DpnII-B | 35.3 | 0.7 | 1.5 |