User:Karmella Haynes/Notebook/BioBrick cloning/2011/07/01

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07/01/11

  • ✓ Cultures/ minipreps: p65 mutagenesis
  • ✓ Assembly: KAH200*, (*Note: DPRE-5xGal4 cloning failed, reassign part number to diff. construct)
  • ✓ DNA denaturing experiment
  • ✓ Sequencing order: SP1A, SP1B



Minipreps
> Check with E/P digests
> Note: old p65 should contain one PstI site, predicted fragments = 470, 313

Reagent Volume Expected:
1,2. p65 re-mut = 783
3. p65 mut (template) = 470, 313
Miniprep digests 7/01/11
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.

--> Failed. Managed to mutate one site but not the other...? Order new primers based on sequencing data from previous p65 mutagenesis. Try both + and - strand.



Assemblies

  1. KAH200: Spacer B433/(E/S dp)/65 ✓ + (1) KAH199/(E/X)/127

> Digests (Fermentas FD)

Reagent Volume   Cloning digest 7/01/11
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. KAH199 (E/X) 0.165 5.01 8.3


> Ligations

Ligation Plate results (lig : neg crtl) 07/02/11
1. Spacer B433(E/S dp)/65, 1 ng + KAH199(E/X)/~3327, 20 ng KAH200 >10:1 (Pick 4)
2. KAH199(E/X)/ 20 ng  
  1 2
Insert DNA 2.5 ---
Vector DNA 2.5 2.5
2x lgn buf (Roche) 6.0 6.0
T4 ligase (NEB) 1.0 1.0
dH2O --- 2.5
  12 μL 12 μL

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



DNA denaturing experiment
--> Concentration of big plasmid DNA appears to fluctuate after freeze-thaw cycles (high, then almost zero, then high again)
--> Faisal: 'Folding of repetitive sequences might affect solubility of transfection plasmids'
--> Test this idea using heat treatment and DMSO on the KAH186-196/MV8 construct
--> See 6/7/11 for previous EcoRI/ApaLI digestion

Reagent Volume Treatment:
1. none
2. 75°C 10 min.
3. 75°C 10 min., 1 μL DMSO
4. 75°C 30 min.
5. 75°C 30 min., 1 μL DMSO
6. 1 μL DMSO
DNA denaturing expt 7/01/11
15 μL/lane; 1% agarose
DNA(plasmid) 2.0 μL
dH2O 9.5 --> Treatment
10X buffer 1.5
EcoRI 1.0
ApaLI 1.0
  15 μL --> 37°C/ ~15 min.

Sequencing order
--> Genewiz, standard premixed
--> f = P0001, r = P0002

  1. SP1A-1, f: good (keep this clone)
  2. SP1A-1, r: good
  3. SP1A-2, f: good
  4. SP1A-2, r: good
  5. SP1B-1, f: good (keep this clone)
  6. SP1B-1, r: good
  7. SP1B-2, f: good
  8. SP1B-2, r: good


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