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From 2012.igem.org

Mark's hair

Mark will get married soon, so he had to spend the entire monday having his hair done. When he finally returned on tuesday, there was no difference.

• 7 September (Kees)

• 5’phosphorylated primers

By amplifying the whole plasmid, including the whole desired insert, only one pcr step is needed which can be used right away to transform bacteria.

The sequence: The Primers:

Legend: Blue: HepBcAg gene Arrows: primers Black: Backbone Red Star: 5’phosphorylation on primers and DNA

The primers used for whole plasmid pcr arrived and were diluted to 100µM. The PCR mixture: component Volume (µL) Primers (FW and rev) 2 Enzyme (Phusion Thermo) 0.5 DMSO 100% 1 Buffer (HF Thermo) 10 Template (20ng/µL) 1 MQ water 34 dNTPs 1

PCR protocol: 1: 98˚C; 2min 2: 98˚C; 30s 3: 63˚C; 30s 4: 72˚C; 30s 15× from step 2 5: 4˚C; ∞ After the PCR, 10µL was used to digest with DpnI, 10µL was mixed with T4 ligase and the rest left untreated. DpnI was heat inactivated after which all three different samples (Ligase, DpnI, normal) were used to transform DH5α.

• 8 September (Kees)

• Colonies

All plates contained colonies, however there was only one on the DpnI-digested plate, which would suggested incomplete digestion rather than a wanted colony. Therefore, more of the sample was plated in order to obtain more colonies.

• 9 September (Kees)

• Checked the plates

There were no new colonies at all on the two new plates.