User:Karmella Haynes/Notebook/BioBrick cloning/2011/08/06

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08/06/11

  • ✓ Minipreps: cultures from yesterday; KAH234 (2), KAH231, 232, 228/MV1
  • ✓ Assembly: KAH235
  • ✓ Retransformations: DPRE (KAH014), MV1 (pcDNA 3.1+)



Minipreps
> Check with E/P digests

Reagent Volume Expected:
1,2. KAH234 = 1041

Vector frag. = 4072
Insert + 1326...
3,4. KAH231/MV1 = 2914
5,6. KAH232/MV1 = 3106
7,8. KAH228 = 3326
File:KAH 080611 gel1.tif
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.

Assemblies

  1. KAH235: (1) DPRE/(E/S)/1740 + (2) KAH234/(E/X)/1041


> Digests (Fermentas FD)

Reagent Volume  
DNA (plasmid) up to 25 μL
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. DPRE (E/S) 1.280 1.91 64.0
2. KAH234 (E/X) 0.465 2.07 23.2


> Ligations

Ligation Plate results (lig : neg crtl) 08/07/11
1. DPRE(E/S)/1740, 22 ng + KAH234(E/X)/~4041, 25 ng KAH235 >10:1 (Pick 2)
2. KAH234(E/X)/ 20 ng  
  1 2
Insert DNA 0.3 ---
Vector DNA 1.0 1.0
2x lgn buf (Roche) 5.0 5.0
T4 ligase (NEB) 1.0 1.0
dH2O 2.7 3.0
  10 μL 10 μL

--> Add to 30 μL DH5α Turbo; plate on 100 μg/mL Amp