Daniel:Notebook/HiResChrPaint/2014-8-19: Difference between revisions

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>Djacobse
(Created page with "=Padlock Probes (Started 8/13/2014)= Back to Calendar ==Ethanol Precipitation ([[Daniel:Notebo...")
 
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===Nanodrop Results===
===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" align="center" valign="bottom"
| width="65" height="15" | Sample
| width="65" | ng/uL (1:5)
| width="65" | ng/uL
| width="65" | ug in 60 uL
| width="65" | ug total
|- style="font-size:12pt"
|style="background-color:#D9D9D9" height="15"  valign="bottom" | PPRL-A
| align="center" align="center" valign="bottom" | 459.9
| align="center" align="center" valign="bottom" | 2,299.5
| align="center" align="center" valign="bottom" | 138.0
| align="center" align="center" valign="bottom" |
|- style="font-size:12pt"
|style="background-color:#D9D9D9" height="15"  valign="bottom" | PPRL-B
| align="center" align="center" valign="bottom" | 465.9
| align="center" align="center" valign="bottom" | 2,329.5
| align="center" align="center" valign="bottom" | 139.8
|style="font-weight:bold" align="center" align="center" valign="bottom" | 277.7
|}


This seems to be a pretty ridiculous amount of probe, but it is consistent with my original nanodrop reading (4822.2 ng/uL of a 20 uL sample, undiluted). I'll push ahead cautiously with a large amount of the probe (but not all).


==Lambda Exonuclease digestion==
==Lambda Exonuclease digestion==
I'll just be using 10 uL of PPRL-A for this one, since it would appear that 10 uL is approximate 20 ug of probe.


'''Reaction Table'''
'''Reaction 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="font-size:12pt;font-weight:bold"
|- style="font-size:12pt;font-weight:bold"
|style="background-color:#C5D9F1" width="117" height="15"  valign="bottom" | Reagent
|style="background-color:#C5D9F1" width="112" height="15"  valign="bottom" | Reagent
|style="background-color:#FCD5B4" width="65" align="center" valign="bottom" | uL
|style="background-color:#FCD5B4" width="65" align="center" valign="bottom" | uL


|- style="font-size:12pt"
|- style="font-size:12pt"
|style="background-color:#C5D9F1" height="15"  valign="bottom" | Amplicon
|style="background-color:#C5D9F1" height="15"  valign="bottom" | Amplicon
| align="center" align="center" valign="bottom" | 30
| align="center" align="center" valign="bottom" | 10
 
|- style="font-size:12pt"
|style="background-color:#C5D9F1" height="15"  valign="bottom" | ug in 10 uL
| align="center" align="center" valign="bottom" | 23.0


|- style="font-size:12pt"
|- style="font-size:12pt"
Line 33: Line 63:


|- style="font-size:12pt"
|- style="font-size:12pt"
|style="background-color:#C5D9F1" height="15"  valign="bottom" | Lambda Exonuclease
|style="background-color:#C5D9F1" height="30"  valign="bottom" | Lambda Exonuclease (5 U/uL)
| align="center" align="center" valign="bottom" | 5
| align="center" align="center" valign="bottom" | 5


|- style="font-size:12pt"
|- style="font-size:12pt"
|style="background-color:#C5D9F1" height="15"  valign="bottom" | nf H2O
|style="background-color:#C5D9F1" height="15"  valign="bottom" | nf H2O
| align="center" align="center" valign="bottom" | 55
| align="center" align="center" valign="bottom" | 75


|- style="font-size:12pt"
|- style="font-size:12pt"
Line 48: Line 78:
#Set up reaction according to table
#Set up reaction according to table
#Incubate at 37C for 1 hour
#Incubate at 37C for 1 hour
#Heat inactivate for 10 minutes at 75C
#Purify using 2 [[Daniel:Protocols/ssDNA Column|ssDNA columns]], elute with 20 uL nfH2O each
#Purify using 2 [[Daniel:Protocols/ssDNA Column|ssDNA columns]], elute with 20 uL nfH2O each


==USER/DpnII Digestion==
===Nanodrop Results===
 
Sample A reported 2.1 ng/uL and sample B reported 1.7 ng/uL.  This means just about everything was lost.
 
==Gel Quantification Check==
 
Based on the results above (no sample after Lambda digestion), I'm going to do a massive gel dilution series to see approximately what the concentration of PPRL-A is.  I'll either repeat for B, or more likely assume it is on the same scale.


'''Reaction 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:#E6B8B7;font-size:12pt;font-weight:bold" align="center"
| width="86" height="15" valign="bottom" | Reagent
| width="86" height="34" | Lane
| width="65" align="center" valign="bottom" | uL
| width="86" | 2 (Ladder)
| align="center" width="65" | 3
| align="center" width="65" | 4
| align="center" width="65" | 5
| align="center" width="65" | 6
| align="center" width="65" | 7
| align="center" width="65" | 8
| align="center" width="65" | 9
| align="center" width="65" | 10


|- style="font-size:12pt"
|- style="font-size:12pt"
|style="background-color:#FCD5B4" height="15"  valign="bottom" | amplicon
|style="background-color:#C5D9F1" height="15"  valign="bottom" | Dilution
| align="center" align="center" valign="bottom" | 35
| align="center" align="center" valign="bottom" | 1
| align="center" align="center" valign="bottom" | 1
| align="center" align="center" valign="bottom" | 1
| align="center" align="center" valign="bottom" | 10
| align="center" align="center" valign="bottom" | 10
| align="center" align="center" valign="bottom" | 10
| align="center" align="center" valign="bottom" | 10
| align="center" align="center" valign="bottom" | 50
| align="center" align="center" valign="bottom" | 50


|- style="font-size:12pt"
|- style="font-size:12pt"
|style="background-color:#FCD5B4" height="15"  valign="bottom" | USER
|style="background-color:#C5D9F1" height="15"  valign="bottom" | uL Sample
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 1
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 1
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 0.5
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 4
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 2
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 1
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 0.5
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 1
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 0.5
 
|- style="font-size:12pt"
|style="background-color:#C5D9F1" height="15"  valign="bottom" | uL TBE
| align="center" align="center" valign="bottom" | 4.5
| align="center" align="center" valign="bottom" | 4.5
| align="center" align="center" valign="bottom" | 5
| align="center" align="center" valign="bottom" | 1.5
| align="center" align="center" valign="bottom" | 3.5
| align="center" align="center" valign="bottom" | 4.5
| align="center" align="center" valign="bottom" | 5
| align="center" align="center" valign="bottom" | 4.5
| align="center" align="center" valign="bottom" | 5
| align="center" align="center" valign="bottom" | 5


|- style="font-size:12pt"
|- style="font-size:12pt"
|style="background-color:#FCD5B4" height="15"  valign="bottom" | nf H2O
|style="background-color:#C5D9F1" height="15"  valign="bottom" | uL buffer
| align="center" align="center" valign="bottom" | 40
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 2
 
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 2
|- style="font-size:12pt;font-weight:bold"
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 2
|style="background-color:#FCD5B4" height="15" valign="bottom" | Total
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 80
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 2
 
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 2
|}
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 2
 
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 2
#Set up reaction according to table in a 0.2 mL PCR tube
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 2
#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="font-size:12pt"
|style="background-color:#FCD5B4" height="15"  valign="bottom" | 10X DpnII Buffer
|style="background-color:#C5D9F1" height="15"  valign="bottom" | Total
| align="center" align="center" valign="bottom" | 10
| align="center" align="center" valign="bottom" | 7.5
| align="center" align="center" valign="bottom" | 7.5
| align="center" align="center" valign="bottom" | 7.5
| align="center" align="center" valign="bottom" | 7.5
| align="center" align="center" valign="bottom" | 7.5
| align="center" align="center" valign="bottom" | 7.5
| align="center" align="center" valign="bottom" | 7.5
| align="center" align="center" valign="bottom" | 7.5
| align="center" align="center" valign="bottom" | 7.5


|- 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:#C5D9F1" height="15"  valign="bottom" | uL Added
| align="center" align="center" valign="bottom" | 5
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 7.2
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 7.2
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 7.2
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 7.2
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 7.2
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 7.2
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 7.2
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 7.2
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 7.2


|- 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:#C5D9F1" height="15"  valign="bottom" | Final uL Added
| align="center" align="center" valign="bottom" | 15
| align="center" align="center" valign="bottom" | 0.96
| align="center" align="center" valign="bottom" | 0.96
| align="center" align="center" valign="bottom" | 0.48
| align="center" align="center" valign="bottom" | 0.384
| align="center" align="center" valign="bottom" | 0.192
| align="center" align="center" valign="bottom" | 0.096
| align="center" align="center" valign="bottom" | 0.048
| align="center" align="center" valign="bottom" | 0.0192
| align="center" align="center" valign="bottom" | 0.0096


|}
|}


#Incubate at 94C for 2 minutes
#Run for 30 minutes at 200 V
#Cool to 37C and incubate for 3 minutes
 
#Add 5 uL DpnII and incubate at 37C for 2 hours
'''Gel Image'''
#Heat inactivate DpnII at 65C for 20 minutes
 
[[Image:2014-08-19-PPRL-A.jpg|500px]]
 
[[Category:HRCP]] [[Category:Lambda Exo]] [[Category:TBE Gel]] [[Category:20140813]]

Latest revision as of 22:41, 9 September 2014

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

Back to Calendar

Ethanol Precipitation (From 8/18/2014)[edit]

  1. Remove from -80C centrifuge at 4400 rpm for 30 minutes at 4C
  2. Add 700 uL 75% EtOH
    1. Break up the pellet and transfer to a 1.5mL tube
  3. Centrifuge once more at 14000 rpm for 10 minutes
  4. Take out supernatant and dry in laminar flow hood for 20 minutes
  5. Resuspend in 20 uL nf H2O

Nanodrop Results[edit]

Sample ng/uL (1:5) ng/uL ug in 60 uL ug total
PPRL-A 459.9 2,299.5 138.0
PPRL-B 465.9 2,329.5 139.8 277.7

This seems to be a pretty ridiculous amount of probe, but it is consistent with my original nanodrop reading (4822.2 ng/uL of a 20 uL sample, undiluted). I'll push ahead cautiously with a large amount of the probe (but not all).

Lambda Exonuclease digestion[edit]

I'll just be using 10 uL of PPRL-A for this one, since it would appear that 10 uL is approximate 20 ug of probe.

Reaction Table

Reagent uL
Amplicon 10
ug in 10 uL 23.0
Lambda Exonuclease Buffer (10X) 10
Lambda Exonuclease (5 U/uL) 5
nf H2O 75
Total 100
  1. Set up reaction according to table
  2. Incubate at 37C for 1 hour
  3. Heat inactivate for 10 minutes at 75C
  4. Purify using 2 ssDNA columns, elute with 20 uL nfH2O each

Nanodrop Results[edit]

Sample A reported 2.1 ng/uL and sample B reported 1.7 ng/uL. This means just about everything was lost.

Gel Quantification Check[edit]

Based on the results above (no sample after Lambda digestion), I'm going to do a massive gel dilution series to see approximately what the concentration of PPRL-A is. I'll either repeat for B, or more likely assume it is on the same scale.

Lane 2 (Ladder) 3 4 5 6 7 8 9 10
Dilution 1 1 1 10 10 10 10 50 50
uL Sample 1 1 0.5 4 2 1 0.5 1 0.5
uL TBE 4.5 4.5 5 1.5 3.5 4.5 5 4.5 5
uL buffer 2 2 2 2 2 2 2 2 2
Total 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5
uL Added 7.2 7.2 7.2 7.2 7.2 7.2 7.2 7.2 7.2
Final uL Added 0.96 0.96 0.48 0.384 0.192 0.096 0.048 0.0192 0.0096
  1. Run for 30 minutes at 200 V

Gel Image

File:2014-08-19-PPRL-A.jpg