User:Karmella Haynes/Notebook/BioBrick cloning/2010/01/24

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01/24/10

  • ✓ Minipreps: KAH140, 141/V0201 (4 each)
  • ✓ PCR colony screen: KAH94, 95/ JDS48, 49 plates

Minipreps
> Check with E/P digests (Oops, cut with E/P instead of XbaI)
--> E/P cuts 94 bp fragment from backbone vector

Reagent Volume Expected:
Empty vector = ~5000
1-4. KAH140/V0201 = ~7945
5-8. KAH141/V0201 = ~7939
E/P digest 1/24/10
15 μL/lane; 1% agarose
DNA(plasmid) 2.0 μL
10X buffer 1.5
EcoRI 1.0
PstI 1.0
dH2O 9.5
  15 μL --> 37°C/ ~15 min.

PCR colony screen
> Use RFP1 primer mix
> Controls: (+) = KAH94 (0.5 μL); (-) = JDS48 (0.5 μL)

Reagent Volume Expected:
100 bp
PCR colony screen 1/24/10
15 μL/lane; 1% agarose
Colony pick 0.0
10 μM primer mix 1.0
2x GoTaq Green 10.0
dH2O 9.0
  20 μL
 
 
 

--> BioRad 96-well: 95°C/ 3 min.; [95°C/ 30 sec., 57°C/ 30 sec., 72°C/ 30 sec.] x30; 72°C/ 3 min.
--> All look positive. Tomorrow check streaks for HS4


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