Daniel:Notebook/HiResChrPaint/2014-9-10: Difference between revisions
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>Djacobse |
>Djacobse |
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(11 intermediate revisions by the same user not shown) | |||
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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=" | | 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" | | | 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. | | 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" | | | 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" | | | 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 43: | Line 100: | ||
#Add 5 uL DpnII and incubate at 37C for 2 hours | #Add 5 uL DpnII and incubate at 37C for 2 hours | ||
#Heat inactivate DpnII at 65C for 20 minutes | #Heat inactivate DpnII at 65C for 20 minutes | ||
#[[Daniel:Protocols/ | |||
===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="15" valign="bottom" | Sample | |||
| 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-weight:bold" height="15" valign="bottom" | ng/uL | |||
| 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-weight:bold" height="15" valign="bottom" | ug total | |||
| align="center" align="center" valign="bottom" | | |||
| align="center" align="center" valign="bottom" | | |||
| align="center" align="center" valign="bottom" | | |||
|style="font-weight:bold" align="center" align="center" valign="bottom" | 24.1 | |||
|} | |||
===TBE Gel=== | |||
#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 | |||
{{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}} | |||
'''Gel Image''' | |||
[[Image:2014-09-09-PPRL.jpg|800px]] | |||
[[Category:HRCP]] [[Category:DpnII]] [[Category:USER]] [[Category:TBE Gel]] [[Category:20140909]] |
Latest revision as of 01:21, 11 September 2014
Padlock Probes II[edit]
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 |
- Set up reaction according to table in a 0.2 mL PCR tube
- Incubate for 2 hours at 37C
- 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[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]
- 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
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