User:Karmella Haynes/Notebook/BioBrick cloning/2011/05/26

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05/26/11

  • ✓ Cultures/minipreps/digests: KAH183/176fwd(6), KAH184/176fwd(6), KAH183/177fwd(6), KAH184/177fwd(6)



Minipreps
> Check with E/ApaI digests
> Note: ApaI site in the vector and in the PEST region of the RFP-Repressor gene

Reagent Volume Expected:
*Vector
Empty KAH176 = 4600*, 1870, 1423
Empty KAH176 = 4600*, 2060, 1423
1-6. KAH183/176fwd = 4600*, 3806, 1870
7-12. KAH184/176fwd = 4600*, 3614, 1870
13-18. KAH183/177fwd = 4600*, 3806, 2063
19-24. KAH184/177fwd = 4600*, 3614, 2063
File:KAH 052611 gel1.tif
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.

--> Observed bands do not match predictions. Recalculate assuming methylated ApaI site in the vector

  • KAH176 = 6421, 1423
  • KAH177 = 6614, 1423
  • 183/176 = 6421, 3806
  • 184/176 = 6421, 3614
  • 183/177 = 6614, 3806
  • 184/177 = 6614, 3614

--> Makes sense, keep positive preps for transfections