User:Karmella Haynes/Notebook/BioBrick cloning/2010/06/01

From OpenWetWare
Jump to: navigation, search
Owwnotebook icon.png Project name Report.pngMain project page
Resultset previous.pngPrevious entry      Next entryResultset next.png

06/01/10

  • ✓ Assemblies: make MV8 and MV3



Assemblies

  1. MV8: MV1/(BsaBI/BstBI)/4605 + Hygro PCR/(BsaBI/BstBI)/1023
  2. MV3: MV7/(BsaBI/BstBI)/3919 + "

> Digests (NEB)
--> Specific notes

Reagent Volume   File:KAH 060210 gel1.tif
30 μL/lane, 1% agarose
DNA (plasmid) 15.0
10x buffer 4 3.0
BsaBI 1.0
BstBI 1.0
dH2O 10.0
 

--> 60°C/ 1 hr.; 65°C/ 1 hr.


> Measure conc.'s

Sample OD260 260/280 ng/μL
1. MV1 (BsaBI/BstBI) 0.636 1.80 31.8
2. MV7 (BsaBI/BstBI) 1.061 1.77 53.0


> Ligations

Ligation Plate results (lig : neg crtl) 06/03/10
1. Hygro PCR(B/B)/1023, 11 ng + MV1(B/B)/4605, 25 ng MV8 1:1 (redo)
2. MV1(B/B)/ 25 ng ~30 colonies
3. Hygro PCR(B/B)/1023, 13 ng + MV7(B/B)/3919, 25 ng MV3 1:1 (redo)
4. MV7(B/B)/ 25 ng ~30 colonies
  1 2 3 4
Insert DNA 1.2 --- 1.5 ---
Vector DNA 0.8 0.8 0.5 0.5
2x lgn buf (Roche) 5.0 5.0 5.0 5.0
T4 ligase (NEB) 1.0 1.0 1.0 1.0
dH2O 2.0 3.2 2.0 3.5
  10 μL 10 μL 10 μL 10 μL

--> R.T./ ~10 min.; Add 50 μL Turbo DH5α