User:Karmella Haynes/Notebook/BioBrick cloning/2011/02/23

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02/23/11
--> mut_SP1A 1 is incorrect; re-order
 * &#x2713; Cultures/ minipreps: G luc (4); 2 mL each (sequence confirm tomorrow)
 * Mutagenesis: activation domains SP1AB 2/3 (mutate PstI site #1); p65 (wait for new primer)

Minipreps > Check with E/P digests

Site-directed Mutagenesis > SP1AB: Mutated PstI sites 2 and 3 last time; still need to mutate site #1 > Stratagene Quick Change mutagenesis kit:
 * 1) SP1AB 2/3, primer mut_SP1A 1
 * 2) p65, primer mut_p65 1

> Templates are ~ 5kb and 4kb

--> BioRad PCR (Block A)
 * 95°C/ 1 min.
 * [95°C/ 1 min., 55°C/ 1 min., 65°C/ 10 min (2 min./kb)] x30
 * 65°C/ 1 min.
 * 4°C/ ∞

(2/24/11) > DpnI Digest (gets rid of methylated template DNA)
 * 1) SP1AB 2/3 mut rxn
 * 2) SP1AB 2/3 control (DNA, 10x buffer, Quick soln. in 25 μL; no PCR)
 * 3) p65 mut rxn
 * 4) p65 control (DNA, 10x buffer, Quick soln. in 25 μL; no PCR)

--> Add 1 μL DpnI enzyme to each sample --> 37°C/ 1 hr. --> Transform 30 μL DH5α Turbo, use 10 μL each sample; Amp plates

2/25/11 --> Results


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