Daniel:Notebook/HiResChrPaint/2013-11-15: Difference between revisions
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>Djacobse (Created page with "=Positive Control (Started 11/14/2013)= Back to Calendar ==Lambda Exonuclease Digestion== 1....") |
>Djacobse |
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4. [[Daniel:Protocols/ssDNA Column|ssDNA column]] purification, elute with 20 uL nfH2O | 4. [[Daniel:Protocols/ssDNA Column|ssDNA column]] purification, elute with 20 uL nfH2O | ||
5. Measure Concentration in Nanodrop | 5. Measure Concentration in Nanodrop | ||
=Positive Control 2 (V6S3 & V6S4)= | |||
==qPCR Amplification== | |||
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" | V6S3 (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" | V6S4 (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=== |
Revision as of 19:37, 15 November 2013
Positive Control (Started 11/14/2013)
Lambda Exonuclease Digestion
1. Reaction table
Sample | V6S1AB | V6S1CD | V6S2AB | V6S2CD |
ng/uL | 128.6 | 125.8 | 147.2 | 150 |
ug in 25 uL | 3.2 | 3.1 | 3.7 | 3.8 |
uL added | 25 | 25 | 25 | 25 |
uL 10x buffer | 5 | 5 | 5 | 5 |
uL Lambda Exonuclease (5U/uL) | 3 | 3 | 3 | 3 |
uL nfH2O | 17 | 17 | 17 | 17 |
total | 50 | 50 | 50 | 50 |
2. Incubate for 1 hour at 37C followed by 10 minute heat inactivation at 75C 3. Pool samples 4. ssDNA column purification, elute with 20 uL nfH2O 5. Measure Concentration in Nanodrop
Positive Control 2 (V6S3 & V6S4)
qPCR Amplification
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 |
V6S3 (12.5x) | 5 | 625 | 5 | 5 | 610 | 1250 |
V6S4 (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