User:Karmella Haynes/Notebook/BioBrick cloning/2011/01/31

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01/31/11

  • ✓ Miniprep digests: p65 w.t.(4), KAH179/176, 180/176, 179/177, 180/177, 179/178, 179/178/MV8 (2 each)



Minipreps
> p65 w.t.: X/P
> Others: E/ApaLI (Note: KAH176, 177, 178 have 2 ApaLI mCherry sites)

Reagent Volume Expected:
1-4. p65 w.t. = 3200, 464, 313, 6

MV8 = 1694, 1243, 1135, 288
Inserts + 234 in last fragment...
5,6. 179/176/MV8 = 4550, 711, 1158
7,8. 180/176/MV8 = 4352, 711, 1158
9,10. 179/177/MV8 = 4550, 711, 1351
11,12. 180/177/MV8 = 4352, 711, 1351
13,14. 179/178/MV8 = 4550, 711, 1362
15,16. 180/178/MV8 = 4352, 711, 1362
File:KAH 013111 gel1.tif
15 μL/lane; 1% agarose
DNA(plasmid) 2.0 μL
10X buffer 1.5
enzyme 1 1.0
enzyme 2 1.0
dH2O 9.5
  15 μL --> 37°C/ ~15 min.


--> Conclusions:

  • p65 cloning worked
  • Unsure about miR sensor assemblies. Largest band shorter than expected and there are many additional bands. Will check restriction sites and send circled clones off for sequencing.

---> mCherry has ApaLI site. Recalculated expected fragments. All make sense except for ~2 kb bands, which are probably partial digests (711+last fragment). Test these by transfection into U2OS.