User:Karmella Haynes/Notebook/BioBrick cloning/2011/08/21

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08/21/11

  • ✓ Cultures/minipreps: KAH223, 232, 231: pick 8 new colonies from old PCR cloning plates
  • ✓ Assembly: KAH235/V0200 (re-do)
  • ✓ Cultures for QIAGEN Plus midipreps: KAH222, 219, 232, 231/pSB31 35 ml cultures (innoculated from 2 ml cultures)



Minipreps
> Check with NcoI digests (p65 has two NcoI sites; expected sizes are for fwd orientation)

Reagent Volume Expected:
Vector
fwd = ~5000
rev = >5000
*Last NcoI frag + 141
1-8. KAH223 = 1031*, 636
9-16. KAH232 = 513, 99, 662*, 636
17-24. KAH231 = 513, 99, 662*, 450
Miniprep digests, 8/21/11
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.

--> Keep circled samples and also 3 more of each for sequencing tomorrow



Assemblies

  1. KAH235/V0200: (1) KAH235/(N/S)/2787 + V0200/(N/S dp)/4442 ✓

> Digests (Fermentas FD)
--> Re-cut KAH235 (N/S); separation last time was not good

Reagent Volume   Cloning digest, 8/21/11
30 μL/lane, 1% agarose
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. KAH235 (N/S) 0.172 1.73 8.6


> Ligations

Ligation Plate results (lig : neg crtl) 08/22/11
1. KAH235(N/S)/2787, 25 ng + V0200(N/S dp)/4442, 20 ng KAH235/V0200 >10:1 (Pick 2)
2. V0200(N/S dp)/ 20 ng  
  1 2
Insert DNA 3.2 ---
Vector DNA 0.8 0.8
2x lgn buf (Roche) 5.0 5.0
T4 ligase (NEB) 1.0 1.0
dH2O --- 3.2
  10 μL 10 μL



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