User:Karmella Haynes/Notebook/BioBrick cloning/2011/06/30

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06/30/11

  • ✓ Minipreps: SP1A (2), SP1B (2), KAH199 (2), KAH200 (2)
  • ✓ Site directed mutagenesis: continue from 6/29/11
  • ✓ Sequencing order: miR sensors set #1 (whole plate failed last time, time try smaller order w/ strip tubes)



Minipreps
> Check SP1A, SP1B, KAH199 with E/P digests
> Check KAH200 with E/S digest

Reagent Volume Expected:
1,2. SP1A KAH018 = 540
3,4. SP1B KAH019 = 840
5,6. KAH199 = 127
7,8. KAH200 = 1839
Miniprep digests 6/30/11
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.

--> KAH200 failed. Try DPRE assembly later with fresh insert. Reassign this number to different construct.
--> Keep 1-4 for sequence confirmation (tomorrow).



Sequencing
--> Primer = DD122 (forward)

  1. 186MV8: good
  2. 184MV8: good
  3. 187MV8: good
  4. 186-196MV8: no priming
  5. 184-196MV8: "
  6. 187-196MV8: "
  7. 186-177MV8: "
  8. 184-177MV8: "
  9. 187-177MV8: "
  10. 186-198MV8: "
  11. 184-198MV8: "
  12. 187-198MV8: "

--> Arggghhh! What is happening to my plasmids?



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