User:Karmella Haynes/Notebook/BioBrick cloning/2010/03/29

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03/29/10

 * &#x2713; Minipreps: KAH145 (4)
 * &#x2713; Assembly: KAH86 (re-make, HSVtkTATA-K missing from original)
 * &#x2713; Site directed mutagenesis (see 01/30/10): DPRE (continue w/ DpnI digest & transformation tomorrow)

Minipreps > Check with E/P/ScaI digests

Assembly
 * 1) KAH86: KAH48/(S/P)/~3280 &#x2713; + (1) KAH81/(X/P)/2640

> Digests (Fermentas FD) --> Digest KAH81 w/ ScaI/X/P

> Measure conc.'s

--> KAH48(S/P) = 33.4 ng/μL

> Ligations

--> R.T./ ~10 min.; Add 50 μL Z-DH5α

Site-directed Mutagenesis > Stratagene Quick Change mutagenesis kit; See 01/30/10 > DPRE contains 2 PstI sites. > Forward primers: mut_DPRE 1, mut_DPRE 2 > Template is ~ 5 kb

--> 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/ ∞

> DpnI Digest of methylated template DNA
 * 1) Forward strand double mutation
 * 2) Neg. control (DNA template, 10x buffer, Quick soln. in 25 μL; no PCR)

--> Add 1 μL DpnI enzyme to each sample --> 37°C/ 1 hr. --> Transform 50 μL z-DH5α, use 15 μL each sample; Amp plates

03/31/10 --> Ratio: >10:1 (20 Fwd strand mut, 0 neg ctrl); Pick 4


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