User:Karmella Haynes/Notebook/BioBrick cloning/2010/03/31

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03/31/10

  • ✓ Minipreps: KAH86 re-built (4)
  • ✓ Assembly: KAH96 re-build
  • ✓ Sequencing (same-day): KAH86 plasmids
  • ✓ Cultures: KAH014 (DPRE) (4)

Minipreps
> Check with E/P digests
> Compare to KAH81

Reagent Volume Expected:
1-4. KAH86 = 2726
5. KAH81 = 2640
E/P digest 03/31/10
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.

--> Samples 1 and 2 appear to run larger than KAH81. Check all 4 via sequencing and proceed with KAH96 assembly.


Assemblies

  1. KAH96: KMC01(5xGal4)/(S/P)/~3300 ✓ + (1) KAH86/(X/P)/2726

> Digests (Fermentas FD)
--> X/P/ScaI triple digest

Reagent Volume   Cloning digest03/31/10
30 μL/lane, 1% agarose -->
DNA (plasmid) 15.0
10x buffer 3.0
XbaI 1.0
PstI 1.0
ScaI 1.0
dH2O 9.0
  30 μL --> 37°C/ ~15 min.


> Measure conc.'s

Sample OD260 260/280 ng/μL
1. KAH94 (X/P) 0.371 2.05 18.5

--> KMC01(S/P) = 62.3 ng/μL


> Ligations

Ligation Plate results (lig : neg crtl) 04/01/10
1. KAH86(X/P)/2726, 41 ng + KMC01(S/P)/~3300, 25 ng KAH96 10:1 (Pick 4)
2. KMC01(S/P)/ 25 ng ~50 colonies
  1 2
Insert DNA 2.5 ---
Vector DNA 0.5 0.5
2x lgn buf (Roche) 5.0 5.0
T4 ligase (NEB) 1.0 1.0
dH2O 1.0 3.5
  10 μL 10 μL

--> R.T./ ~10 min.; Add 50 μL Z-DH5α


Sequencing (Genewiz, same-day, 04/01/10)
--> Confirm HSVtkTATA-K

  1. KAH86-1 P0001: good
  2. KAH86-2 P0001: good
  3. KAH86-3 P0001: good
  4. KAH86-4 P0001: good



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