User:Karmella Haynes/Notebook/BioBrick cloning/2010/01/30

{| width="800"
 * style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] Project name
 * style="background-color: #F2F2F2" align="center"|  |Main project page
 * style="background-color: #F2F2F2" align="center"|  |Main project page


 * colspan="2"|
 * colspan="2"|

01/30/10

 * &#x2713; Minipreps: KAH139/V0201
 * &#x2713; Site directed mutagenesis: EF-1α promoter

Minipreps > Check with XbaI digest

Site-directed Mutagenesis > EF-1α promoter contains 2 PstI sites. For quick-change mutagenesis same-strand primers are used to generate single-stranded plasmids for transformation of E. coli. (This actually works???) > Stratagene Quick Change mutagenesis kit: Try forward and reverse strand mutagenesis (alhtough one is sufficient), since I have both sets of primers on hand... > Template is ~ 6kb
 * 1) Forward primers: EF-1a Pst1f, EF-1a Pst2f
 * 2) Reverse primers: EF-1a Pst1r, EF-1a Pst2r

--> BioRad PCR (Block A)
 * 95°C/ 1 min.
 * [95°C/ 1 min., 55°C/ 1 min., 65°C/ 12 min (2 min./kb)] x30
 * 65°C/ 1 min.
 * 4°C/ ∞

> DpnI Digest (gets rid of methylated template DNA)
 * 1) Forward strand double mutation
 * 2) Reverse strand double mutation
 * 3) 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

02/02/10 --> Ratios look great. ~10 colonies on #1 & 2; 0 colonies on #3 (neg. ctrl)


 * }