Difference between revisions of "User:Karmella Haynes/Notebook/BioBrick cloning/2013/03/06"

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* Streaked single colonies on gridded plates yesterday
 
* Streaked single colonies on gridded plates yesterday
 
* Used bit of streak for PCR today
 
* Used bit of streak for PCR today
 +
* All parts are on pSB1A3-based vectors (http://partsregistry.org/Part:pSB1A3)
 +
** Use primers VF2 and VR to amplify inserts
  
 
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
 
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
Line 20: Line 22:
 
| bgcolor=#cfcfcf | Reagent
 
| bgcolor=#cfcfcf | Reagent
 
| bgcolor=#cfcfcf | Volume  
 
| bgcolor=#cfcfcf | Volume  
| rowspan="7" | <u>Expected:</u><br>1. BB 1 = size<br>2. BB2 = size<br>
+
| rowspan="7" | <u>Expected:</u><br>Primers add ~250bp<br>1-3. K084007 (835 bp) = 1085<br>4-6. R0071 (53 bp) = 303<br>7. K081016 (735 bp) = 985
| rowspan="7" | [[Image:KAH030713_gel1.jpg|250px|PCR gel]]<br>10 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
+
| rowspan="7" | [[Image:KAH030713_gel1.jpg|300px|PCR gel]]<br>10 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
 
|-
 
|-
 
| Bacteria from tip || ---
 
| Bacteria from tip || ---

Latest revision as of 18:18, 7 March 2013

Owwnotebook icon.pngKarmella's BioBrick Cloning <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
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03/06/13

  • Colony PCR: for Rene's project



Colony PCR

  • Streaked single colonies on gridded plates yesterday
  • Used bit of streak for PCR today
  • All parts are on pSB1A3-based vectors (http://partsregistry.org/Part:pSB1A3)
    • Use primers VF2 and VR to amplify inserts
Reagent Volume Expected:
Primers add ~250bp
1-3. K084007 (835 bp) = 1085
4-6. R0071 (53 bp) = 303
7. K081016 (735 bp) = 985
PCR gel
10 μL/lane; 1% agarose; Ladder
Bacteria from tip ---
10 μM VF2 primer 1.0
10 μM VR primer 1.0
2x GoTaq Green 12.5
dH2O 10.5
  25.0

PCR

  • 95°C, 3 min
  • [95°C, 15 sec; 57°C 15 sec; 72 °C 60 sec] x35
  • 72°C, 3 min
  • 4°C, ∞