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From 2012.igem.org

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Revision as of 08:39, 13 September 2012

Hepatitis B outside modification

• 27 August (Kees)

• Whole plasmid PCR

Instead of using the general FW and Rev primers, the whole plasmid can be amplified alternatively. This allows a more controlled Tm and only 1 amplicon instead of 2 which have to be combined. The same steps 1-4 are still required.

First try with Pfusion polymerase did not have any result.

• 29 August (Kees)

• Gradient PCR

A greadient pcr was performed using Taq polymerase. This can only determine wether the primers do work at a certain, yet for phusion undertermined temperature. Besides, Taq adds an A, causing a mutation in the final step. Therefore, the products can not be used, even if we accept the absence of proofreading in Taq.

The bands on the gel are as expected somewhat over 3k bp. This proofs this pcr step is possible.

• Phusion Polymerase PCR

After the proof was given that this first pcr step is possible, we use phusion to obtain usable product. The new annealing temperature is 63˚C, based upon the Fermentas calculation. The elongation time was set to 2min, which should allow elongation of the product at an elongation speed of 1k bp/min. Again, the pcr did not work. Therefore, new primers were ordered which can execute the whole pcr at once, including a TEV-protease sight.