User:Karmella Haynes/Notebook/BioBrick cloning/2010/02/02

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02/02/10

 * &#x2713; Colony PCR: from EF-1α site directed mutagenesis plates, PCR w/ BioBrick primers
 * &#x2713; Assembly: EF-1α into V0120

Colony PCR & BioBricking > Pick colonies from site directed mutagenesis plates from 2/01 (6 each) > Use Forward EF1a/ Reverse EF1a (contain Xba and Pst ends) > Cut products with X/P (PstI leaves desired mutants in tact and cuts non-mutated fragments) > Isolate 1184 bp band for cloning

PCR 1-6. Forward strand mutagenesis clones 7-12. Reverse strand mutagenesis clones 13. EF-1α (original template plasmid DNA)

--> BioRad 96-well --> Zymo clean & concentrate; elute w/ 20 μL dH2O
 * 95°C/ 10 min.
 * [95°C/ 30 sec., 57°C/ 30 sec., 72°C/ 1 min.] x30
 * 72°C/ 3 min.
 * 4°C/ ∞

Digest --> Digest samples w/ XbaI/PstI

--> Gel purify for Assemblies

Assemblies
 * 1) EF-1α(1): &#x2713; EF-1α PCR 1/(X/P)/1184 + &#x2713; V0120/(X/P)/3200
 * 2) EF-1α(2): &#x2713; EF-1α PCR 2/(X/P)/1184 + "
 * 3) EF-1α(3): &#x2713; EF-1α PCR 3/(X/P)/1184 + "

> Measure conc.'s

> Ligations


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