User:Karmella Haynes/Notebook/BioBrick cloning/2010/01/13

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01/13/10

  • ✓ Sequencing order: KAH126-133/MV2 transfection plasmids (primers DD122B, DD123B)
  • ✓ Minipreps: KAH117/MV2 (4)
  • ✓ Cultures: KAH94, 95 re-transformed (2 each); KAH117/MV2 (6) KAH118/MV2 (6)
  • ✓ Assemblies: KAH137, 138

Minipreps
> Check with E/P digests

Reagent Volume Expected:
1-4. KAH117/MV2 = 7758
E/P digests 1/13/10
15 μL/lane; 1% agarose
DNA(plasmid) 5.0
10X buffer 1.5
EcoRI 1.0
PstI 1.0
dH2O 6.5
  15 μL --> 37°C/ ~15 min.

--> None are correct. Re-do digestion of inserts (KAH117 & 118) and ligation (into MV2) if recent attempt fails.


Assemblies

  1. KAH137: (1) KAH135/(E/S)/2353 + (3) NLSstop2(KAH010)/(E/X)/3200
  2. KAH138: (2) KAH136/(E/S)/1941 + "

> Digests (Fermentas FD)

Reagent Volume   Cloning digests 1/13/10
30 μL/lane, 1% agarose
DNA (plasmid) 10.0
10x buffer 3.0
enzyme 1 1.0
enzyme 2 1.0
dH2O 15.0
  30 μL --> 37°C/ ~30 min.


> Measure conc.'s

Sample OD260 260/280 ng/μL
1. KAH135(E/S) 0.308 2.01 15.4
2. KAH136(E/S) 0.380 1.78 19.0
3. KAH010(E/X) 0.502 1.99 25.1


> Ligations

Ligation Plate results (lig : neg crtl) 01/14/10
1. KAH135(E/S)/2353, 29 ng + KAH010(E/X)/3200, 20 ng KAH137 10:1 (Pick 2)
2. KAH136(E/S)/1941, 24 ng + " KAH138 10:1 (Pick 2)
3. KAH010(E/X)/ 20 ng  
  1 2 3
Insert DNA 1.8 1.3 ---
Vector DNA 0.8 0.8 0.8
2x lgn buf (Roche) 5.0 5.0 5.0
T4 ligase (NEB) 1.0 1.0 1.0
dH2O 1.4 1.9 4.2
  10μL 10μL 10μL --> R.T./~10 min.; Add 30 μL Z-DH5α



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