User:Karmella Haynes/Notebook/BioBrick cloning/2011/01/11

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01/11/11

  • ✓ Assemblies: KAH171-173 (re-build miR sensor constructs, might be a problem with old polyA, DDB82)
  • ✓ Transformation: MI127 (for minipreps)



Assemblies
> Re-build miR sensor using new polyA (from Mara, MI127) and miRNA or siRNA luc RNAi target sites (from Joe)
> Re-build RFP repressor construct for testing new polyA (no HS4 insulators)

  1. KAH171: (1) JDS46/(S/P)/~3387 + (3) MI127/(X/P)/225
  2. KAH172: (2) JDS47/(S/P)/~3398 + "
  3. KAH173: HPK/(E/S)/516 ✓ + KAH73/(E/X)/~5638 ✓

> Digests (Fermentas FD)

Reagent Volume   1/11/11 cloning digest
30 μL/lane, 1% agarose
DNA (plasmid) up to 25 μL
10x buffer 3.0
enzyme 1 1.0
enzyme 2 1.0
dH2O ---
  30 μL --> 37°C/ ~30 min.


> Measure conc.'s

Sample OD260 260/280 ng/μL
1. JDS46 (S/P) 0.275 1.79 13.77
2. JDS47 (S/P) 0.291 1.87 14.55
3. MI127 (X/P) 0.104 1.84 5.22


> Ligations

Ligation Plate results (lig : neg crtl) 1/12/11
1. MI127(X/P)/225, 3.3 ng + JDS46(S/P)/~3387, 25 ng KAH171 >10:1 (Pick 2)
2. JDS46(S/P)/ 25 ng  
3. MI127(X/P)/225, 3.3 ng + JDS47(S/P)/~3398, 25 ng KAH172 >10:1 (Pick 2)
4. JDS47(S/P)/ 25 ng  
5. HPK(E/S)/516, 4.6 ng + KAH73(E/X)/~5638, 25 ng KAH173 2:1 (Pick 6)
6. KAH73(E/X)/ 25 ng  
  1 2 3 4 5 6
Insert DNA 1.0 --- 1.0 --- 0.5 ---
Vector DNA 2.0 2.0 2.0 2.0 1.0 1.0
2x lgn buf (Roche) 5.0 5.0 5.0 5.0 5.0 5.0
T4 ligase (NEB) 1.0 1.0 1.0 1.0 1.0 1.0
dH2O 1.0 2.0 1.0 2.0 2.5 3.0
  10 μL 10 μL 10 μL 10 μL 10 μL 10 μL

--> 10 min./ R.T.; add to 30 μL DH5α turbo



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