User:Karmella Haynes/Notebook/BioBrick cloning/2009/11/25

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11/25/09

  • ✓ Minipreps: KAH94(4), 95(4)
  • ✓ Cultures: KAH96(2), 97(2), 98(2), 011(6)
  • ✓ Make 10x P1 buffer
  • ✓ Check KAH011 & 012 sequences

Minipreps
> Check with E/P digests

Reagent Volume Expected:
1. KAH94 = 5011
2. KAH95 = 5093
 E/P digest 11/25/09
DNA(plasmid) 2.0 μL
10X buffer 1.5
EcoRI 1.0
PstI 1.0
dH2O 9.5
  15 μL --> 37°C/ ~15 min.

Buffer P1 (Qiagen)
> 50 mM Tris·Cl pH 8, 10 mM EDTA, 10 μg/mL RNAse A
> 10x stock P1:

Reagent Final conc. Vol
Tris·Cl pH 8 500 mM 125 mL 250 mL
EDTA 100 mM 50 mL 100 mL
dH2O
   75 mL 150 mL
    250 mL 500 mL

> Autoclave
> To use, dilute 10x to 1x with sterile dH2O; add RNAse A to 10 μg/mL; store at 4°C

Sequence results

  1. KAH011-1-P0001: bad clone
  2. KAH011-2-P0001: bad clone
  3. KAH012-1-P0001: good (keep this one)
  4. KAH012-2-P0001: good

> Pick 6 clones from KAH011 plate for new minipreps, then sequence


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