Toronto/11 August 2010

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Running PCR for PET-expression vector. Primers arrived yesterday

-->using 1uL of stock DNA for E1-->E5b

10x reaction buffer 2.5uL

ddH2O 17.15uL

Fwd primer 0.2uL

Rvs primer 0.2uL

dNTPs 2mM 3.75uL

DNA(E1-->E5b) 1uL

Taq Polymerase 0.2uL

6 tubes for E1-E5b

1tube for -ve control

-Ran Program “PCR” under file “Anish”

-Re-running transformation of E1-E5b to RosDE3 cells. 1uL DNA into 100uL of cells

→ PUC19 +ve control → 3uL (RosDE3 cells)

-Followed protocol for Transformation

+ve &E1 & E2 plated 10uL and 50uL

plated 5uL and 25uL from E3 on

-Left in 37C incubator

PCR products for pET expression vector DNA (E1-E5b) good yield >100ng/uL & 1.9+(260/280) ratio

-Ready to test enzyme expession in E. Coli!!!!!