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From 2012.igem.org

Hepatitis B outside modification

• 10 September (Kees)

• Colony PCR

The colonies were picked after which part of it was put in pcr-tubes for checking the content and part pas plated for continuation of the colony.

All the bands have a similar size of about 1100bp. The expected construct should contain a fragment of 1250bp. Since this is not the case and all the inserts are of the same size, the pcr failed.

• 11 September (Kees)

• Reverse PCR coil modification Same was done as on 7 September, with the following modifications: - 25 pcr cycles instead of 15. This will yield more template, taking the risk of modifications. - Analyse pcr result on a agarose gel before further processing. - Ligate overnight at 16 degrees if gel shows linear band at about 3kb. (protocols: http://www.neb.com/nebecomm/products_intl/faqproductM0202.asp, http://www.idtdna.com/pages/docs/user-guides-and-protocols/mutagenesis-application-guide.pdf, p.32) - Digest with DpnI after ligation

• 12 September (Kees)

• Digestion and plating After digestion of the ligated sample and the non-ligated sample, the cells were transformed: - Template (positive control) - Ligated + digested - Digested only

• 13 September (Kees)

One colony grew on the DpnI+Ligated plate. This colony will be checked with colony pcr and inocculated to produce anything that is in there.