Daniel:Notebook/HiResChrPaint/2014-9-10: Difference between revisions

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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:#B8CCE4;font-size:12pt;font-weight:bold"
| width="65" height="30"  valign="bottom" | Sample
| width="65" align="center" | 1:1 ng/uL
| width="65" align="center" | 5:1 ng/uL
| width="65" align="center" | 5:1 ng/uL Projected
| width="65" align="center" | 10:1 ng/uL
| width="65" align="center" | 10:1 ng/uL Projected
| width="65" align="center" | Average ng/uL
| width="65" align="center" | Standard Deviation
| width="65" align="center" | ug in 100 uL
|- style="font-size:12pt"
|style="font-weight:bold" height="15" valign="bottom" | PPRL-A
| align="center" align="center" valign="bottom" | 1944.1
| align="center" align="center" valign="bottom" | 373
| align="center" align="center" valign="bottom" | 1865
| align="center" align="center" valign="bottom" | 184.4
| align="center" align="center" valign="bottom" | 1844
| align="center" align="center" valign="bottom" | 1884.4
| align="center" align="center" valign="bottom" | 52.8
|style="font-weight:bold" align="center" align="center" valign="bottom" | 188.4
|- style="background-color:#D9D9D9;font-size:12pt"
|style="font-weight:bold" height="15" valign="bottom" | PPRL-B
| align="center" align="center" valign="bottom" | 2064.8
| align="center" align="center" valign="bottom" | 421.7
| align="center" align="center" valign="bottom" | 2108.5
| align="center" align="center" valign="bottom" | 203
| align="center" align="center" valign="bottom" | 2030
| align="center" align="center" valign="bottom" | 2067.8
| align="center" align="center" valign="bottom" | 39.3
|style="font-weight:bold" align="center" align="center" valign="bottom" | 206.8
|}
Usually after ethanol precipitation I get really high results, so I'll just say these are fine and continue as though I have a reasonable amount of material.


==DpnII/USER Digestion==
==DpnII/USER Digestion==
Line 9: Line 47:


'''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="background-color:#B8CCE4;font-size:12pt;font-weight:bold" align="center"
|- style="background-color:#B8CCE4;font-size:12pt;font-weight:bold" align="center"
| width="93" height="33" | Reagent
| width="65" height="30" | Reagent
| width="65" | PPRL (uL)
| width="71" | PPRL-A1 (uL)
| width="71" | PPRL-A2 (uL)
| width="71" | PPRL-B1 (uL)
| width="65" | PPRL-B2 (uL)


|- style="font-size:12pt"
|- style="font-size:12pt"
|style="background-color:#FCD5B4" height="15"  valign="bottom" | amplicon
|style="background-color:#FCD5B4" height="15"  valign="bottom" | amplicon
| align="center" align="center" valign="bottom" | 30
| align="center" align="center" valign="bottom" | 50
| align="center" align="center" valign="bottom" | 50
| 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" | ug Input
|style="background-color:#FCD5B4" height="15"  valign="bottom" | ug Input
| align="center" align="center" valign="bottom" | 2.0
| align="center" align="center" valign="bottom" | 94.2
| align="center" align="center" valign="bottom" | 94.2
| align="center" align="center" valign="bottom" | 103.4
| align="center" align="center" valign="bottom" | 103.4


|- style="font-size:12pt"
|- style="font-size:12pt"
|style="background-color:#FCD5B4" height="15"  valign="bottom" | USER
|style="background-color:#FCD5B4" height="15"  valign="bottom" | USER
| align="center" align="center" valign="bottom" | 5
| align="center" align="center" valign="bottom" | 5
| 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"
|- style="font-size:12pt"
|style="background-color:#FCD5B4" height="15"  valign="bottom" | nf H2O
|style="background-color:#FCD5B4" height="15"  valign="bottom" | nf H2O
| align="center" align="center" valign="bottom" | 45
| align="center" align="center" valign="bottom" | 30
| align="center" align="center" valign="bottom" | 30
| align="center" align="center" valign="bottom" | 30
| align="center" align="center" valign="bottom" | 30


|- 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" | 80
| align="center" align="center" valign="bottom" | 85
| align="center" align="center" valign="bottom" | 85
| align="center" align="center" valign="bottom" | 85
| align="center" align="center" valign="bottom" | 85


|}
|}
Line 38: Line 95:
#Set up reaction according to table in a 0.2 mL PCR tube
#Set up reaction according to table in a 0.2 mL PCR tube
#Incubate for 2 hours at 37C
#Incubate for 2 hours at 37C
#Add 15 uL according to DpnII Primer table (below)
#Add 10 uL DpnII buffer (10X)
#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
#[[Daniel:Protocols/
 
===Nanodrop Results===


'''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;font-weight:bold" align="center"
|- style="background-color:#B8CCE4;font-size:12pt;font-weight:bold"
| width="93" height="31" | Reagent
| width="65" height="15"  valign="bottom" | Sample
| width="65" | PPRL (uL)
| width="65" align="center" valign="bottom" | PPRL-A1
| width="65" align="center" valign="bottom" | PPRL-A2
| width="65" align="center" valign="bottom" | PPRL-B1
| width="65" align="center" valign="bottom" | PPRL-B2


|- style="font-size:12pt"
|- style="font-size:12pt"
|style="background-color:#FCD5B4" height="15"  valign="bottom" | 10X DpnII Buffer
|style="font-weight:bold" height="15"  valign="bottom" | ng/uL
| align="center" align="center" valign="bottom" | 10
| align="center" align="center" valign="bottom" | 154.1
| align="center" align="center" valign="bottom" | 131
| align="center" align="center" valign="bottom" | 153
| align="center" align="center" valign="bottom" | 163.7
 
|- style="background-color:#D9D9D9;font-size:12pt"
|style="font-weight:bold" height="15"  valign="bottom" | ug in 40 uL
| align="center" align="center" valign="bottom" | 6.2
| align="center" align="center" valign="bottom" | 5.2
| align="center" align="center" valign="bottom" | 6.1
| align="center" align="center" valign="bottom" | 6.5


|- style="font-size:12pt"
|- style="font-size:12pt"
|style="background-color:#FCD5B4" height="30"  valign="bottom" | 100 uM RE-DpnII-V4 guide oligo
|style="font-weight:bold" height="15"  valign="bottom" | ug total
| align="center" align="center" valign="bottom" | 5
| align="center" align="center" valign="bottom" | &nbsp;
 
| align="center" align="center" valign="bottom" | &nbsp;
|- style="font-size:12pt;font-weight:bold"
| align="center" align="center" valign="bottom" | &nbsp;
|style="background-color:#FCD5B4" height="15" valign="bottom" | Total
|style="font-weight:bold" align="center" align="center" valign="bottom" | 24.1
| align="center" align="center" valign="bottom" | 15


|}
|}


#Incubate at 94C for 2 minutes
===TBE Gel===
#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===
#For each sample, 5 uL TBE, 2 uL 6x dye, 1 uL sample; Load 7.5 uL each
#Run gel at 220V for 25 minutes


{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
{{Template:GelLanes|Lane2=Low Mass Ladder|Lane3=PPRL-A, pre-digest 10:1|Lane4=PPRL-A, pre-digest 40:1|Lane5=PPRL-A1|Lane6=PPRL-A2|Lane7=PPRL-B, pre-digest 10:1|Lane8=PPRL-B, pre-digest 40:1|Lane9=PPRL-B1|Lane10=PPRL-B2}}
|- 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"
'''Gel Image'''
|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"
[[Image:2014-09-09-PPRL.jpg|800px]]
|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


|}
[[Category:HRCP]] [[Category:DpnII]] [[Category:USER]] [[Category:TBE Gel]] [[Category:20140909]]

Latest revision as of 01:21, 11 September 2014

Padlock Probes II[edit]

Back to Calendar

Nanodrop Results[edit]

Sample 1:1 ng/uL 5:1 ng/uL 5:1 ng/uL Projected 10:1 ng/uL 10:1 ng/uL Projected Average ng/uL Standard Deviation ug in 100 uL
PPRL-A 1944.1 373 1865 184.4 1844 1884.4 52.8 188.4
PPRL-B 2064.8 421.7 2108.5 203 2030 2067.8 39.3 206.8

Usually after ethanol precipitation I get really high results, so I'll just say these are fine and continue as though I have a reasonable amount of material.

DpnII/USER Digestion[edit]

Reaction Table

Reagent PPRL-A1 (uL) PPRL-A2 (uL) PPRL-B1 (uL) PPRL-B2 (uL)
amplicon 50 50 50 50
ug Input 94.2 94.2 103.4 103.4
USER 5 5 5 5
nf H2O 30 30 30 30
Total 85 85 85 85
  1. Set up reaction according to table in a 0.2 mL PCR tube
  2. Incubate for 2 hours at 37C
  3. Add 10 uL DpnII buffer (10X)
  4. Incubate at 94C for 2 minutes
  5. Cool to 37C and incubate for 3 minutes
  6. Add 5 uL DpnII and incubate at 37C for 2 hours
  7. Heat inactivate DpnII at 65C for 20 minutes
  8. [[Daniel:Protocols/

Nanodrop Results[edit]

Sample PPRL-A1 PPRL-A2 PPRL-B1 PPRL-B2
ng/uL 154.1 131 153 163.7
ug in 40 uL 6.2 5.2 6.1 6.5
ug total       24.1

TBE Gel[edit]

  1. For each sample, 5 uL TBE, 2 uL 6x dye, 1 uL sample; Load 7.5 uL each
  2. Run gel at 220V for 25 minutes


Lane 1 2 3 4 5 6 7 8 9 10 11 12
Sample Low Mass Ladder PPRL-A, pre-digest 10:1 PPRL-A, pre-digest 40:1 PPRL-A1 PPRL-A2 PPRL-B, pre-digest 10:1 PPRL-B, pre-digest 40:1 PPRL-B1 PPRL-B2

Gel Image

File:2014-09-09-PPRL.jpg