Daniel:Notebook/ComboLock/2016-11-1: Difference between revisions
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>Djacobse |
>Djacobse |
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<li>Rolling Circle Amplification (Samples 1,3,5,7)</li> | <li>Rolling Circle Amplification (Samples 1,3,5,7)</li> | ||
<ol type="A"> | <ol type="A"> | ||
<li>Prepare samples in new tube according to following table</li> | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#92CDDC;font-size:12pt;font-weight:bold" align="center" | |||
| width="160" height="30" | Reagent | |||
| width="85" | uL Added | |||
| width="85" | Master Mix (8.1X) | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Template | |||
| align="center" align="center" valign="bottom" | 5 | |||
| align="center" valign="bottom" | NA | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | RCA Primer (100 uM) | |||
| align="center" align="center" valign="bottom" | 2.5 | |||
| align="center" align="center" valign="bottom" | 20.25 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | dNTP (1 mM) | |||
| align="center" align="center" valign="bottom" | 0.8 | |||
| align="center" align="center" valign="bottom" | 6.48 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | 10X Buffer | |||
| align="center" align="center" valign="bottom" | 2 | |||
| align="center" align="center" valign="bottom" | 16.2 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Phi29 | |||
| align="center" align="center" valign="bottom" | 1 | |||
| align="center" align="center" valign="bottom" | 8.1 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | nfH2O | |||
| align="center" align="center" valign="bottom" | 8.7 | |||
| align="center" align="center" valign="bottom" | 70.47 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Total | |||
|style="font-weight:bold" align="center" align="center" valign="bottom" | 20 | |||
|style="font-weight:bold" align="center" align="center" valign="bottom" | 121.5 | |||
|} | |||
<li>qPCR</li> | <li>qPCR</li> |
Revision as of 17:59, 1 November 2016
Circularization Optimization Testing
Sample Matrix
Sample | Polymerase | Incubation Time | RCA Y/N |
Sample 1 (AB) | Klentaq | ON | Y |
Sample 2 (AB) | Klentaq | ON | N |
Sample 3 (AB) | Klentaq | 2 hr | Y |
Sample 4 (AB) | Klentaq | 2 hr | N |
Sample 5 (AB) | Phusion | ON | Y |
Sample 6 (AB) | Phusion | ON | N |
Sample 7 (AB) | Phusion | 2 hr | Y |
Sample 8 (AB) | Phusion | 2 hr | N |
Protocol
- Circularization (all samples)
- Heat inactivate enzyme by incubating for 2 minutes at 94C
- Exonuclease Digestion
- Prepare exonuclease I/III mix by mixing 30 uL exonuclease I (20 units/μL) and 6 uL exonuclease III (100 units/μL) in 1:1 ratio
- Add 2 μL of exonuclease I/III mix to reaction
- Mix the reaction by swirling pipette around the well 5 times
- Incubate reaction at 37 ºC for 2 hours
- Heat inactivate enzyme by incubating at 94C for 5 minutes
- Rolling Circle Amplification (Samples 1,3,5,7)
- Prepare samples in new tube according to following table
- qPCR
- Set up reaction according to table below
- Make master mixes according to following recipes
- 54.25 uL nfH2O
- 77.5 uL SYBR Fast
- 1.55 uL AmpF
- Add 43 uL master mix to each well
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x24
- 72C 2 min
- 16C hold
- TBE Gel
- Mix 48 uL TBE, 12 uL 6x loading dye
- Aliquot 10 uL per sample/ladder lane onto parafilm
- Add 2 uL of sample or ladder to correct drop
- Load 10 uL in to well
- Run gel for 22 minutes at 250V
- Open gel and stain with 3 uL SYBR Gold for 3 minutes
- Rinse gel and image in gel doc
Reagent | uL Added | Master Mix (8.1X) |
Template | 5 | NA |
RCA Primer (100 uM) | 2.5 | 20.25 |
dNTP (1 mM) | 0.8 | 6.48 |
10X Buffer | 2 | 16.2 |
Phi29 | 1 | 8.1 |
nfH2O | 8.7 | 70.47 |
Total | 20 | 121.5 |