Primers working!!!

So I am wrangling with getting these literature based PKS I primers working for my PhD project. Finally success!!!

PKS I Primers

degKS2F 5’-GCIATGGAYCCICARCARMGIVT 

degKS2R 5’- GTICCIGTICCRTGISCYTCIAC

760 bp fragment

Schirmer, A., R. Gadkari, C. D. Reeves, F. Ibrahim, E. F. DeLong, and C. R. Hutchinson. 2005. Metagenomic analysis reveals diverse polyketide synthase gene clusters in microorganisms associated with the marine sponge Discodermia dissoluta. Appl Environ Microbiol 71:4840-4849.

Master mix for one reaction (bring up to 15 uL total with DNA and water)

in uL

H2O 6.00

PCR Buffer 1.5

DNTP 1.5

BAS 0.14

Primers 0.15 each

Taq 0.1

MgCl 2.4

DMSO 1.4 ***I typically don’t use DMSO so swap in water here)

GEL!!!! The first well in each line is the positive control

Image

 

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