User:Karmella Haynes/Notebook/BioBrick cloning/2010/03/21

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03/21/10

  • ✓ Assembly: KAH142/V0201



Assembly

  1. KAH142/V0201: (1) KAH142/(N/S)/4571 + V0201/(N/S dp)/5157 ✓


> Digest (Fermentas FD)
--> Re-do KAH142 N/S digest

Reagent Volume   N/S digest 3/21/10
30 μL/lane, 1% agarose
DNA (plasmid) 10.0
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. KAH142 (N/S) 0.927 1.93 46.3

> Ligations

Ligation Plate results (lig : neg crtl) 03/22/10
1. KAH142(N/S)/4571, 44 ng + V0201(N/S dp)/5157, 25 ng KAH142/V0201 >10:1 (Pick 4)
2. V0201(N/S dp)/ 25 ng 10 colonies
  1 2
Insert DNA 1.0 ---
Vector DNA 1.0 1.0
2x lgn buf (Roche) 5.0 5.0
T4 ligase (NEB) 1.0 1.0
dH2O 2.0 3.0
  10 μL 10 μL

--> R.T./ ~10 min., Add to 50 μL Z-DH5α



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