Daniel:Notebook/HiResChrPaint/2014-8-20: Difference between revisions

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'''DpnII Primer Table'''
'''DpnII Primer 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:#B8CCE4;font-size:12pt"
|- style="background-color:#B8CCE4;font-size:12pt;font-weight:bold" align="center"
| width="91" height="15"  valign="bottom" | Reagent
| width="93" height="31" | Reagent
| width="65" align="center" valign="bottom" | uL
| width="65" | PPRL-A (uL)
| width="65" | PPRL-B (uL)


|- style="font-size:12pt"
|- style="font-size:12pt"
|style="background-color:#FCD5B4" height="15"  valign="bottom" | 10X DpnII Buffer
|style="background-color:#FCD5B4" height="15"  valign="bottom" | 10X DpnII Buffer
| align="center" align="center" valign="bottom" | 10
| align="center" align="center" valign="bottom" | 10
| align="center" align="center" valign="bottom" | 10


|- style="font-size:12pt"
|- style="font-size:12pt"
|style="background-color:#FCD5B4" height="30"  valign="bottom" | 100 uM RE-DpnII-V4 guide oligo
|style="background-color:#FCD5B4" height="30"  valign="bottom" | 100 uM RE-DpnII-V4 guide oligo
| align="center" align="center" valign="bottom" | 5
| align="center" align="center" valign="bottom" | 5
| align="center" align="center" valign="bottom" | 5


|- style="font-size:12pt;font-weight:bold"
|- style="font-size:12pt;font-weight:bold"
|style="background-color:#FCD5B4" height="15"  valign="bottom" | Total
|style="background-color:#FCD5B4" height="15"  valign="bottom" | Total
| align="center" align="center" valign="bottom" | 15
| align="center" align="center" valign="bottom" | 15
| align="center" align="center" valign="bottom" | 15



Revision as of 18:12, 20 August 2014

Padlock Probes (Started 8/13/2014)

Back to Calendar

Lambda Exonuclease Digestion

Since it looks like I might have less DNA than I originally thought (makes sense, several hundred ug is pretty unlikely), I'm going to run the lambda exonuclease digestion on the remaining padlock probes, A and B.

Reaction Table

Reagent PPRL-A (uL) PPRL-B (uL)
Amplicon 45 55
ug Input 20.7 25.6
Lambda Exo Buffer (10X) 10 10
Lambda Exonuclease (5 U/uL) 5 5
nf H2O 40 30
Total 100 100
  1. Set up reaction according to table
  2. Incubate at 37C for 2 hours
  3. Heat inactivate for 10 minutes at 75C
  4. Purify using ssDNA columns, elute with 20 uL nfH2O

Nanodrop Results

Sample ng/uL ug in 20 uL Input (ug) Efficiency (%) ug Total
PPRL-A 132.8 2.7 20.7 12.83
PPRL-B 214 4.3 25.6 16.72 6.9

This result is good enough to proceed with.

USER/DpnII Digestion

Reaction Table

Reagent PPRL-A (uL) PPRL-B (uL)
amplicon 15 15
ug Input 2.0 3.2
USER 5 5
nf H2O 60 60
Total 80 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-A (uL) PPRL-B (uL)
10X DpnII Buffer 10 10
100 uM RE-DpnII-V4 guide oligo 5 5
Total 15 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