User:Karmella Haynes/Notebook/BioBrick cloning/2013/03/06

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(03/06/13)
Current revision (20:18, 7 March 2013) (view source)
(03/06/13)
 
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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 -->
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| 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 || ---

Current revision

Karmella's BioBrick Cloning 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, ∞


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