User:Karmella Haynes/Notebook/BioBrick cloning/2010/03/19

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03/19/10

  • ✓ Minipreps: KAH142, 96/V0201 (2 each)
  • ✓ Sequencing order: KAH96/V0201
  • ✓ Cultures: KAH142/V0201 (8)

Minipreps
> Check with XbaI digest
> XbaI fragment will have an extra 641 bp from vector sequence; remaining vector = 4516

Reagent Volume Expected:
1. KAH142/V0201 = 4571 (5212)
2. KAH96/V0201 = 2825 (3466)
File:KAH 031910 gel1.tif
15 μL/lane; 1% agarose
DNA(plasmid) 2.0 μL
10X buffer 1.5
EcoRI 1.0
dH2O 10.5
  15 μL --> 37°C/ ~15 min.

--> Sequence KAH96/V0201 plasmids
--> Pick 8 new colonies for KAH146/V0201



Sequencing order

  1. KAH96_V0201-1 3T3: HSVtkTATA-Kozak is missing
  2. KAH96_V0201-1 BGHR: good
  3. KAH96_V0201-2 3T3: HSVtkTATA-Kozak is missing
  4. KAH96_V0201-2 BGHR: good

--> Minimal promoter is missing! KAH142 might also be bad. Sequence construct intermediates KAH86 and 96 in V0120.