User:Karmella Haynes/Notebook/BioBrick cloning/2010/03/17

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03/17/10

  • ✓ Minipreps: KAH142 (6)
  • ✓ Assembly: KAH142 & 96 into V0201

Minipreps
> Check with E/S digests

Reagent Volume Expected:
1-6. KAH142 = 4571
E/S digest 03/17/10
15 μL/lane; 1% agarose
DNA(plasmid) 2.0 μL
10X buffer 1.5
EcoRI 1.0
SpeI 1.0
dH2O 9.5
  15 μL --> 37°C/ ~15 min.

Assemblies

  1. KAH142/V0201: (1) KAH142/(N/S)/4571 + V0201/(N/S dp)/5157
  2. KAH96/V0201: (2) KAH96/(N/S/)/2825 + "

> Digests (Fermentas FD)
--> Use ApaLI to cut V0120 in KAH96 digest (V0120 expected = 1246, 776, 745, 497)

Reagent KAH142 KAH96 Hover name
30 μL/lane, 1% agarose
DNA (plasmid) 10.0 10.0
10x buffer 3.0 3.0
NotI 1.0 1.0
SpeI 1.0 1.0
ApaLI --- 1.0
dH2O 15.0 14.0
  30 μL 30 μL --> 37°C/ ~30 min.


> Measure conc.'s

Sample OD260 260/280 ng/μL
1. KAH142 (N/S) 0.972 1.87 48.6
2. KAH96 (N/S) 0.867 1.80 43.3


> Ligations

Ligation Plate results (lig : neg crtl) 03/18/10
1. KAH142(N/S)/4571, > 44 ng + V0201(N/S dp)/5157, 25 ng KAH142/V0201 >10:1 (Pick 2)
2. KAH96(N/S)/2825, > 27 ng + " KAH96/V0201 4:1 (Pick 2)
2. V0201(N/S dp)/ 25 ng 4 colonies
  1 2 3
Insert DNA 2.0 2.0 ---
Vector DNA 1.0 1.0 1.0
2x lgn buf (Roche) 5.0 5.0 5.0
T4 ligase (NEB) 1.0 1.0 1.0
dH2O 1.0 1.0 3.0
  10 μL 10 μL 10 μL

--> R.T./ ~10 min., Add to 30 μL Z-DH5α


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