User:Jingyang Chen/Notebook/Severe Malaria Protein Production/2008/07/30

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Cloning of PF10_0160 (soluble portion)

Examined the results of the latest sequencing results of submitted clones. None of them had the right sequence. Many of them had insertions and deletions in poly A regions.

I should probably use a higher fidelity DNA polymerase to PCR amplify the target gene.

Use PfuTurbo from Stratagene.

1 uL of F/R primer mix 2.5 uL 10X PfuTurbo Buffer 1 uL 3D7 strain cDNA template (around 26 ng) 0.625 uL dNTPs (10 mm each, from Roche) 1 uL (2.5 U) PfuTurbo DNA polymerase 18.9 uL water

PCR cycling conditions

Use previous two step PCR, except

Denaturing temperature raised to 95 C, extended to 30 s during the cycles.

Annealing: extend intracycle time to 30 s