User:Karmella Haynes/Notebook/BioBrick cloning/2011/08/22

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08/22/11

  • ✓ Cultures/minipreps: KAH235/V0200 (2)
  • ✓ Midipreps: Pc-TF/pSB31 constructs for Manching



Minipreps
> Check with N/S digests

Reagent Volume Expected:
1,2. KAH235/V0200 = 4442, 2787
Image:KAH 082211 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.



Midipreps
> Use QIAGEN Midiprep Plus kit > Check with NcoI digests

Reagent Volume Expected:
1. KAH235/V0200 = size
Image:KAH 082211 gel2.tif
2 μL DNA, 15 μL/lane; 1% agarose

Midiprep digests, repeat
5 μL DNA, 15 μL/lane; 1% agarose
DNA(plasmid) 2.0 / 5.0 μL
10X buffer 1.5
NcoI 1.0
dH2O 10.5 / 7.5
  15 μL --> 37°C/ ~15 min.

--> Nanodrop reading showed 150 - 270 ng/μL for these preps, but no DNA is visible on the gel. Try re-transformation, followed by midi prep.



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