User:Karmella Haynes/Notebook/BioBrick cloning/2011/01/24

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01/24/11

 * &#x2713; Minipreps: SP1AB w.t. (6), KAH160, 165, 170/pSB31 (6 each)
 * &#x2713; Mutagenesis: site directed mutagenesis for SP1AB w.t.
 * &#x2713; Cultures: KAH160 (10); 2 mL

Minipreps > SP1AB w.t.: X/P > Others: X/NcoI (BioBrick constructs contain 2 NcoI sites, vector has 1)

--> Conclusions: pSB31 does not have an NcoI site. NcoI should generate a short internal band in the BioBrick clones. Only KAH170 worked. Pick more colonies for KAH160 and 165 (10 each).

Site-directed Mutagenesis > SP1AB has 3 PstI sites > Stratagene Quick Change mutagenesis kit: Try all three primers at once --> Forward primers: mut_SP1A 1, mut_SP1B 1, mut_SP1B 2

> Template is ~ 5kb

--> 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/ ∞

(1/25/11) > DpnI Digest (gets rid of methylated template DNA)
 * 1) Forward strand triple mutation
 * 2) 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 z-DH5α, use 10 μL each sample; Amp plates

01/26/11 --> Results: Great ratio (one colony on neg ctrl.). Pick several and check by restriction digest.


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