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

Back to Calendar

Nanodrop Results

DpnII/USER Digestion

Reaction Table

Reagent PPRL (uL)
amplicon 30
ug Input 2.0
USER 5
nf H2O 45
Total 80
  1. Set up reaction according to table in a 0.2 mL PCR tube
  2. Incubate for 2 hours at 37C
  3. 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
  1. Incubate at 94C for 2 minutes
  2. Cool to 37C and incubate for 3 minutes
  3. Add 5 uL DpnII and incubate at 37C for 2 hours
  4. 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