Daniel:Notebook/ComboLock/2016-11-1: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Djacobse
(Created page with "=Circularization Optimization Testing= Back to Calendar ===Sample Matrix=== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:He...")
 
>Djacobse
Line 64: Line 64:
==Protocol==
==Protocol==


<ol start="2">
<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 20 uL exonuclease I (20 units/μL) and 4 uL exonuclease III (100 units/μL) in 1:1 ratio</li>
<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

Back to Calendar

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

  1. Circularization (all samples)
    1. Heat inactivate enzyme by incubating for 2 minutes at 94C
  2. Exonuclease Digestion
    1. 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
    2. Add 2 μL of exonuclease I/III mix to reaction
    3. Mix the reaction by swirling pipette around the well 5 times
    4. Incubate reaction at 37 ºC for 2 hours
    5. Heat inactivate enzyme by incubating at 94C for 5 minutes
  3. Rolling Circle Amplification (Samples 1,3,5,7)
    1. qPCR
      1. Set up reaction according to table below
      2. Make master mixes according to following recipes
        1. 54.25 uL nfH2O
        2. 77.5 uL SYBR Fast
        3. 1.55 uL AmpF
      3. Add 43 uL master mix to each well
      4. qPCR Cycles
        1. 95C 3 min
        2. 95C 3 sec
        3. 55C 30 sec
        4. 72C 20 sec
        5. plate read
        6. goto b x24
        7. 72C 2 min
        8. 16C hold
    2. TBE Gel
      1. Mix 48 uL TBE, 12 uL 6x loading dye
      2. Aliquot 10 uL per sample/ladder lane onto parafilm
      3. Add 2 uL of sample or ladder to correct drop
      4. Load 10 uL in to well
      5. Run gel for 22 minutes at 250V
      6. Open gel and stain with 3 uL SYBR Gold for 3 minutes
      7. Rinse gel and image in gel doc

    Results