User:Karmella Haynes/Notebook/BioBrick cloning/2011/07/05

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07/05/11

  • ✓ Cultures/minipreps: KAH201, 202, 203 (3 each)
  • ✓ Sequencing order: KAH201, 202, 203
  • ✓ Retransform: mCherry E3000
  • ✓ (Re-) Assembly: miR sensor 183-177/MV8 (as a control for Daniel's set)
  • ✓ Order oligos: new mutagenesis primers for p65



Minipreps
> Check with E/P digests

Reagent Volume Expected:
1-3. KAH201 = 226
4-6. KAH202 = 325
7-9. KAH203 = 155
Miniprep digest 7/05/11
15 μL/lane; 1% agarose
DNA(plasmid) 3.0 μL
10X buffer 1.5
EcoRI 1.0
PstI 1.0
dH2O 8.5
  15 μL --> 37°C/ ~15 min.


Sequencing order
--> Send all samples for Genewiz sequencing
--> Use P0001 (fwd) primer for all samples

  1. KAH201-1: good (keep this clone)
  2. KAH201-2: good
  3. KAH201-3: good
  4. KAH202-1: good (keep this clone)
  5. KAH202-2: good
  6. KAH202-3: good
  7. KAH203-1: no priming
  8. KAH203-2: good (keep this clone)
  9. KAH203-3: good

Note: B433 spacer is actually 42 bp (2 repeat segments, not three)



Assembly
--> Not sure about previous miR sensor construct vectors; start re-building and check with ApaLI instead of E/ApaLI --> Note: Use KAH177/MV8 E/X digest from 1/25/11, not 5/25/11

  1. KAH183-177/MV8: KAH183/(E/S)/2383 ✓ + KAH177/MV8/(E/X)/3384 ✓


> Ligations

Ligation Plate results (lig : neg crtl) 07/06/11
1. KAH183(E/S)/2383, 15 ng + KAH177/MV8(E/X)/7978, 25 ng KAH183-177/MV8 >10:1 (Pick 4)
2. vector(c/d)/ 25 ng  
  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


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



Order oligos
--> Previous attempts to mutate the second PstI site failed. Order new primers and try both strands...

  1. p65 mut 2 (+ strand): TGCTGCAACTGCAaTTTGATGATGAAG
  2. p65 mut 3 (- strand): CTTCATCATCAAAtTGCAGTTGCAGCA




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