Daniel:Notebook/ComboLock/2016-11-1: Difference between revisions
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>Djacobse (Created page with "=Circularization Optimization Testing= Back to Calendar ===Sample Matrix=== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:He...") |
>Djacobse |
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==Protocol== | ==Protocol== | ||
<ol start=" | <ol start="4"> | ||
<li>Circularization (all samples)</li> | <li>Circularization (all samples)</li> | ||
<ol type="A"> | <ol type="A"> | ||
Line 70: | Line 70: | ||
<li>Exonuclease Digestion</li> | <li>Exonuclease Digestion</li> | ||
<ol type="A"> | <ol type="A"> | ||
<li>Prepare exonuclease I/III mix by mixing | <li>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</li> | ||
<li>Add 2 μL of exonuclease I/III mix to reaction</li> | <li>Add 2 μL of exonuclease I/III mix to reaction</li> | ||
<li>Mix the reaction by swirling pipette around the well 5 times</li> | <li>Mix the reaction by swirling pipette around the well 5 times</li> | ||
Line 76: | Line 76: | ||
<li>Heat inactivate enzyme by incubating at 94C for 5 minutes</li> | <li>Heat inactivate enzyme by incubating at 94C for 5 minutes</li> | ||
</ol> | </ol> | ||
<li>Rolling Circle Amplification (Samples 1,3,5,7)</li> | |||
<ol type="A"> | |||
<li>qPCR</li> | <li>qPCR</li> | ||
<ol type="A"> | <ol type="A"> | ||
<li>Set up reaction according to table below</li> | <li>Set up reaction according to table below</li> | ||
<li>Make master mixes according to following recipes</li> | |||
<ol type="a"> | |||
<li>54.25 uL nfH2O</li> | |||
<li>77.5 uL SYBR Fast</li> | |||
<li>1.55 uL AmpF</li> | |||
</ol> | |||
<li>Add 43 uL master mix to each well</li> | |||
<li>qPCR Cycles</li> | |||
<ol type="a"> | |||
<li>95C 3 min</li> | |||
<li>95C 3 sec</li> | |||
<li>55C 30 sec</li> | |||
<li>72C 20 sec</li> | |||
<li>plate read</li> | |||
<li>goto b x24</li> | |||
<li>72C 2 min</li> | |||
<li>16C hold</li> | |||
</ol></ol> | |||
<li>TBE Gel</li> | |||
<ol type="A"> | |||
<li>Mix 48 uL TBE, 12 uL 6x loading dye</li> | |||
<li>Aliquot 10 uL per sample/ladder lane onto parafilm</li> | |||
<li>Add 2 uL of sample or ladder to correct drop</li> | |||
<li>Load 10 uL in to well</li> | |||
<li>Run gel for 22 minutes at 250V</li> | |||
<li>Open gel and stain with 3 uL SYBR Gold for 3 minutes</li> | |||
<li>Rinse gel and image in gel doc</li> | |||
</ol></ol> | |||
===Results=== | |||
<gallery perrow=2 heights=300px widths=300px> | |||
File:|qPCR curve | |||
File:|Gel image | |||
</gallery> |
Revision as of 16:43, 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)
- 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