Daniel:Notebook/ComboLock/2017-3-6: Difference between revisions

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>Djacobse
(Created page with "=Padlock0601 Test (Started Wed March 1)= Back to Calendar After reading some papers I'm going to give it...")
 
>Djacobse
Line 10: Line 10:
<li>Maximal Rolling Circle Amplification</li>
<li>Maximal Rolling Circle Amplification</li>
<ol type="A">
<ol type="A">
<li>
<li>Create master mix according to following table</li>
<li>Add 15 uL master mix to each tube</li>
 
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="background-color:#8DB4E2;font-size:12pt;font-weight:bold" align="center"
| width="180" height="30" | Reagent
| width="85" | Single Rxn Vol
| width="85" | 16.2X MM uL
 
|- style="font-size:12pt"
| height="15"  valign="bottom" | Template
| align="center" align="center" valign="bottom" | 4
| align="center" align="center" valign="bottom" | 0
 
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | RCA Hexamer (1 mM)
| align="center" align="center" valign="bottom" | 0.5
| align="center" align="center" valign="bottom" | 8.1
 
|- style="font-size:12pt"
| height="15"  valign="bottom" | dNTP (1 mM)
| align="center" align="center" valign="bottom" | 5
| align="center" align="center" valign="bottom" | 81
 
|- 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" | 32.4
 
|- 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" | 6.48
 
|- style="font-size:12pt"
| height="15"  valign="bottom" | nfH2O
| align="center" align="center" valign="bottom" | 7.1
| align="center" align="center" valign="bottom" | 115.02
 
|- 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" | 243
 
|}
 
<li>Add 16 uL master mix to each tube</li>
<li>Add 4 uL template according to table</li>
<li>Add 4 uL template according to table</li>
<li>Incubate at 95 C for 5 minutes</li>
<li>Incubate at 95 C for 5 minutes</li>
Line 20: Line 69:
<li>Heat kill enzyme by incubating 10 minutes at 65C; Hold at 12C</li>
<li>Heat kill enzyme by incubating 10 minutes at 65C; Hold at 12C</li>
</ol></ol>
</ol></ol>
[[Category:ComboLock]] [[Category:20170301]]

Revision as of 00:16, 7 March 2017

Padlock0601 Test (Started Wed March 1)

Back to Calendar

After reading some papers I'm going to give it one more go to see if I can't amplify the DNA from last week better. I'll use the method of largest amplification I know: most time using random hexamers as primers.

Protocol

  1. Maximal Rolling Circle Amplification
    1. Create master mix according to following table
    2. Reagent Single Rxn Vol 16.2X MM uL
      Template 4 0
      RCA Hexamer (1 mM) 0.5 8.1
      dNTP (1 mM) 5 81
      10X Buffer 2 32.4
      Phi29 1 0
      BSA (10 mg/mL) 0.4 6.48
      nfH2O 7.1 115.02
      Total 20 243
    3. Add 16 uL master mix to each tube
    4. Add 4 uL template according to table
    5. Incubate at 95 C for 5 minutes
    6. Lower to 55C for 15 minutes
    7. Lower to 30C for at least 2 minutes
    8. Add 1 uL Phi29 polymerase to each sample
    9. Incubate 15 hours at 30C
    10. Heat kill enzyme by incubating 10 minutes at 65C; Hold at 12C