Daniel:Notebook/HiResChrPaint/2013-10-22: Difference between revisions
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>Djacobse |
>Djacobse |
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1. Reaction Table | 1. Reaction Table | ||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="font-size:12pt" align="center" | |||
|style="background-color:#FFFF00" align="center" width="65" height="45" | | |||
| width="65" | 10 nM OligoPool | |||
| width="65" | 2X Kapa SYBR qPCR MM | |||
| width="65" | 100 uM AP1V61U-Amino | |||
| width="65" | 100 uM AP2V6 | |||
| width="65" | H20 | |||
| width="65" | Total Volume | |||
|- style="background-color:#8DB4E2;font-size:12pt" align="center" | |||
| height="15" | per rxn | |||
| align="center" | 0.4 | |||
| align="center" | 50 | |||
| align="center" | 0.4 | |||
| align="center" | 0.4 | |||
| align="center" | 48.8 | |||
| align="center" | 100 | |||
|- style="font-size:12pt" align="center" | |||
| height="30" | BSPP 1 (12.5x) | |||
| align="center" | 5 | |||
| align="center" | 625 | |||
| align="center" | 5 | |||
| align="center" | 5 | |||
| align="center" | 610 | |||
| align="center" | 1250 | |||
|- style="font-size:12pt" align="center" | |||
| height="30" | BSPP 2 (12.5x) | |||
| align="center" | 5 | |||
| align="center" | 625 | |||
| align="center" | 5 | |||
| align="center" | 5 | |||
| align="center" | 610 | |||
| align="center" | 1250 | |||
|} | |||
2. Aliquot 100 uL into each of 12 qPCR tubes | |||
3. PCR protocol: | |||
i. PCR 3 min 95C | |||
ii. 95C 3 sec | |||
iii. 55C 30 sec | |||
iv. 72C 20 sec | |||
v. plate read | |||
vi. goto ii x17 | |||
vii. 72C 2 min | |||
viii. 16C hold | |||
4. Pooled 3 samples together each (300 uL each, 4 pools per set, 8 total) | |||
5. [[Daniel:Protocols/Qiaquick_Column|Column Purification]], elute with 40 uL EB | |||
6. Measure concentrations in nanodrop | |||
===qPCR Results=== | |||
[[Image:2013-10-22-BSPPControl.png|600px]] | |||
===Nanodrop Results=== | |||
{| 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" align="center" | |- style="font-size:12pt;font-weight:bold" align="center" | ||
| width="65" height="15" | | | width="65" height="15" | Probe | ||
| width="65" | BSPP 1A | | width="65" | BSPP 1A | ||
| width="65" | BSPP 1B | | width="65" | BSPP 1B | ||
Line 35: | Line 94: | ||
|- style="font-size:12pt" align="center" | |- style="font-size:12pt" align="center" | ||
|style="font-weight:bold" height="15" | ug in | |style="font-weight:bold" height="15" | ug in 40 uL | ||
| align="center" | 3. | | align="center" | 3.9 | ||
| align="center" | 3.8 | |||
| align="center" | 3.9 | |||
| align="center" | 3.7 | |||
| align="center" | 3.6 | |||
| align="center" | 3.7 | |||
| align="center" | 3.6 | |||
| align="center" | 3.3 | | align="center" | 3.3 | ||
|- style="font-size:12pt" align="center" | |- style="font-size:12pt" align="center" | ||
|style="font-weight:bold" height="15" | Total | |style="font-weight:bold" height="15" | Total | ||
| align="center" | | | align="center" | | ||
| align="center" | | | align="center" | | ||
| align="center" | | | align="center" | | ||
| align="center" | | | align="center" | 15.2 | ||
| align="center" | | | align="center" | | ||
| align="center" | | | align="center" | | ||
| align="center" | | | align="center" | | ||
| align="center" | | | align="center" | 14.2 | ||
|} | |} | ||
==DpnII Digestion== |
Revision as of 01:09, 23 October 2013
BSPP Control
- For first production of BSPP control, see 09/03/2013
Since I'm out of control group, I'm going to make some more.
qPCR
1. Reaction Table
10 nM OligoPool | 2X Kapa SYBR qPCR MM | 100 uM AP1V61U-Amino | 100 uM AP2V6 | H20 | Total Volume | |
per rxn | 0.4 | 50 | 0.4 | 0.4 | 48.8 | 100 |
BSPP 1 (12.5x) | 5 | 625 | 5 | 5 | 610 | 1250 |
BSPP 2 (12.5x) | 5 | 625 | 5 | 5 | 610 | 1250 |
2. Aliquot 100 uL into each of 12 qPCR tubes 3. PCR protocol: i. PCR 3 min 95C ii. 95C 3 sec iii. 55C 30 sec iv. 72C 20 sec v. plate read vi. goto ii x17 vii. 72C 2 min viii. 16C hold 4. Pooled 3 samples together each (300 uL each, 4 pools per set, 8 total) 5. Column Purification, elute with 40 uL EB 6. Measure concentrations in nanodrop
qPCR Results
File:2013-10-22-BSPPControl.png
Nanodrop Results
Probe | BSPP 1A | BSPP 1B | BSPP 1C | BSPP 1D | BSPP 2A | BSPP 2B | BSPP 2C | BSPP 2D |
ng/uL | 97.9 | 94.5 | 96.9 | 91.9 | 90.2 | 92.5 | 90.7 | 82.4 |
ug in 40 uL | 3.9 | 3.8 | 3.9 | 3.7 | 3.6 | 3.7 | 3.6 | 3.3 |
Total | 15.2 | 14.2 |