Daniel:Notebook/ComboLock/2017-2-3: Difference between revisions

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<li>Prepare 7X Master mix according to table below</li>
<li>Prepare 7X Master mix according to table below</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="155" height="30" | Reagent
| width="65" | uL Added
| width="65" | Master Mix (8.2X)


<li>Add 15 uL master mix to each tube</li>
|- 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 (10 uM)
| align="center" align="center" valign="bottom" | 2.5
| align="center" align="center" valign="bottom" | 20.5
 
|- style="font-size:12pt"
| height="15"  valign="bottom" | dNTP (1 mM)
| align="center" align="center" valign="bottom" | 5
| align="center" align="center" valign="bottom" | 41
 
|- 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" | 16.4
 
|- style="font-size:12pt"
| height="15"  valign="bottom" | Phi29
| 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" | BSA (10 mg/mL)
| align="center" align="center" valign="bottom" | 0.4
| align="center" align="center" valign="bottom" | 3.28
 
|- style="font-size:12pt"
| height="15"  valign="bottom" | nfH2O
| align="center" align="center" valign="bottom" | 4.1
| align="center" align="center" valign="bottom" | 33.62
 
|- style="background-color:#BFBFBF;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" | 131.2
 
|}
 
<li>Add 14 uL master mix to each tube</li>
<li>Add sample to new reaction tubes</li>
<li>Add sample to new reaction tubes</li>
<li>Incubate at 37C for 3 hours</li>
<li>Incubate at 37C for 3 hours</li>
<li>Incubate at 65C for 10 minutes;Hold at 10C until next step</li></ol>
<li>Incubate at 65C for 10 minutes;Hold at 10C until next step</li></ol>

Revision as of 17:17, 3 February 2017

Version 2 Oligos Round 2 (Started yesterday)

Back to Calendar

Protocol-Part 4-RCA

  1. Exonuclease Digestion
    1. Mix 15 uL Exo I (20U/uL) and 3 uL ExoIII (100U/uL)
    2. Add 2 uL to each sample, mix by swirling pipette tip
    3. Incubate for 1 hour at 37C
    4. Heat kill by incubating for 5 min at 95C
  2. Rolling Circle Amplification
    1. Prepare 7X Master mix according to table below
    2. Reagent uL Added Master Mix (8.2X)
      Template 5 NA
      RCA Primer (10 uM) 2.5 20.5
      dNTP (1 mM) 5 41
      10X Buffer 2 16.4
      Phi29 1 0
      BSA (10 mg/mL) 0.4 3.28
      nfH2O 4.1 33.62
      Total 20 131.2
    3. Add 14 uL master mix to each tube
    4. Add sample to new reaction tubes
    5. Incubate at 37C for 3 hours
    6. Incubate at 65C for 10 minutes;Hold at 10C until next step