Daniel:Notebook/ComboLock/2017-4-29: Difference between revisions

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>Djacobse
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<li>Add 15 uL master mix to each tube</li>
<li>Add 15 uL master mix to each tube</li>
<ol type="a"><li>Samples "A-C" get + master mixes; samples "D-F" get - master mixes</li></ol>
<li>Add 4 uL sample to appropriate tubes</li>
<li>Add 1 uM RCA primer and to new reaction tubes according to following rules</li>
<ol type="a">
<li>"A,D" samples get 2 uL each LLRC & primer12RC </li>
<li>"B,E" samples get 2 uL each LLRC*** & primer12RC </li>
<li>"C,F" samples get 4 uL LLRC***</li></ol>
<li>Heat reactions up to 95C for 5 min</li>
<li>Heat reactions up to 95C for 5 min</li>
<li>Cool to 55C and incubate for 15 minutes</li>
<li>Cool to 55C and incubate for 15 minutes</li>
<li>Cool to 30C and add enzyme</li>
<li>Cool to 30C and add 1 uL enzyme</li>
<li>Incubate for 3 hours at 30C</li>
<li>Incubate for 3 hours at 30C</li>
<li>Incubate for 65 minutes at 20C to denature</li>
<li>Incubate for 65 minutes at 20C to denature</li>
</ol>
</ol>
<li>qPCR</li>
<ol>
<li>Bglii digestion</li>
<ol type="A">
<ol type="A">
<li>Make qPCR master mixes according to following recipe</li>
<li>Make the following reactions; '''Do not add Bglii yet'''</li>
 
{| 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="background-color:#8DB4E2" width="145" height="30" | Reagent
|style="background-color:#8DB4E2" width="85" | Single Rxn Vol
|style="background-color:#95B3D7" width="85" | Master Mix (16.2X)
 
|- style="font-size:12pt"
| height="15"  valign="bottom" | RCA Rxn
| align="center" align="center" valign="bottom" | 7
| align="center" align="center" valign="bottom" | 0
 
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | 10X Buffer 3.1
| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 32.4
 
|- style="font-size:12pt"
| height="15"  valign="bottom" | Bglii
| align="center" align="center" valign="bottom" | 1
| align="center" align="center" valign="bottom" | 0
 
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | nfH2O
| align="center" align="center" valign="bottom" | 10
| align="center" align="center" valign="bottom" | 162
 
|- 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" | 194.4
 
|}
 
<li>Aliquot 12 uL master mix into new tube</li>
<li>Add 7 uL appropriate sample</li>
<li>Mix with vortexer, spin down, and add 1 uL Bglii</li>
<li>Incubate 2 hr 37C</li>
<li>Heat kill with 65C for 20 min</li>
<li>Hold at 12C</ol>
 




[[Category:ComboLock]] [[Category:20170429]]
[[Category:ComboLock]] [[Category:20170429]]

Revision as of 00:08, 30 April 2017

Production Run (Started Yesterday)

Back to Calendar

Protocol-Part 4

  1. Exonuclease Digestion
    1. Mix 17.5 uL Exo I (20U/uL) and 3.5 uL ExoIII (100U/uL)
    2. Add 2 uL to each sample, mix by swirling pipette tip
    3. Incubate for 2 hours at 37C
    4. Heat kill by incubating for 5 min at 95C
  2. Rolling Circle Amplification
    1. Prepare master mix according to table below
    2. Reagent Single Rxn (uL) Master Mix X (10.2X) Master Mix Y (6.2X)
      Enzyme NA NEB Thermo
      Sample 4 0 0
      p12RC*** (10 uM) 0.2 2.04 1.24
      RCA Primer LLRC*** (10 uM) 0.2 2.04 1.24
      dNTP (10 mM) 0.5 5.1 3.1
      10X Buffer 2 20.4 12.4
      Phi29 1 0 0
      BSA (10 mg/mL) 0.4 4.08 0
      nfH2O 11.7 119.34 75.02
      Total 20 153 93
    3. Add 15 uL master mix to each tube
    4. Add 4 uL sample to appropriate tubes
    5. Heat reactions up to 95C for 5 min
    6. Cool to 55C and incubate for 15 minutes
    7. Cool to 30C and add 1 uL enzyme
    8. Incubate for 3 hours at 30C
    9. Incubate for 65 minutes at 20C to denature
    1. Bglii digestion
      1. Make the following reactions; Do not add Bglii yet
      2. Reagent Single Rxn Vol Master Mix (16.2X)
        RCA Rxn 7 0
        10X Buffer 3.1 2 32.4
        Bglii 1 0
        nfH2O 10 162
        Total 20 194.4
      3. Aliquot 12 uL master mix into new tube
      4. Add 7 uL appropriate sample
      5. Mix with vortexer, spin down, and add 1 uL Bglii
      6. Incubate 2 hr 37C
      7. Heat kill with 65C for 20 min
      8. Hold at 12C