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

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07/01/11

 * &#x2713; Cultures/ minipreps: p65 mutagenesis
 * &#x2713; Assembly: KAH200*, (*Note: DPRE-5xGal4 cloning failed, reassign part number to diff. construct)
 * &#x2713; DNA denaturing experiment
 * &#x2713; Sequencing order: SP1A, SP1B

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

--> 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 &#x2713; + (1) KAH199/(E/X)/127

> Digests (Fermentas FD)

> Measure conc.'s

> Ligations

--> 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

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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