User:Karmella Haynes/Notebook/BioBrick cloning/2010/08/04

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08/04/10

  • ✓ Cultures/ minipreps: KAH160-170 (2 each, 2 mL)
  • ✓ Sequencing order: KAH015 (TetO-FUW-Oct4 + microMCS)



Minipreps
> Check with E/P digests

Reagent Volume Expected:
1,2. KAH160 = 1147
3,4. KAH161 = 1147
5,6. KAH162 = 1147
7,8. KAH163 = 2131
9,10. KAH164 = 2134
11,12. KAH165 = 991
13,14. KAH166 = 991
15,16. KAH167 = 991
17,18. KAH168 = 1955
19,20. KAH169 = 1958
21,22. KAH170 = 955
Digest check 8/04/10
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.



Sequencing order
> KAH015 (TetO-FUW-Oct4 + microMCS)
--> Forward = KAH015seq F
--> Reverse = KAH015seq R

  1. KAH015-1 F: low quality seq (early termination)
  2. KAH015-2 F: low quality seq (high background)
  3. KAH015-3 F: 2x microMCS inserted in reverse orientation
  4. KAH015-4 F: good
  5. KAH015-1 R: low quality seq (early termination)
  6. KAH015-2 R: low quality seq (high background)
  7. KAH015-3 R: 2x microMCS inserted in reverse orientation
  8. KAH015-4 R: good

--> Keep and use KAH015-4


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