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Team:Wageningen UR/Journal/week20
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{{Template:WUR}} | {{Template:WUR}} | ||
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| + | '''Hepatitis B general''' | ||
| + | |||
| + | ---- | ||
| + | |||
| + | * 2nd try colony PCR of the transformations from 4. September | ||
| + | -> one colony seems to have the correct insert in the pSB1C3 backbone | ||
| + | -> no conclusion can be made about samples containing the Hep-inside coil fragment in the Bba_J04500 brick | ||
'''Hepatitis B outside modification''' | '''Hepatitis B outside modification''' | ||
| + | |||
| + | ---- | ||
| + | |||
* 10 September (Kees) | * 10 September (Kees) | ||
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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. | 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. | ||
| + | |||
| + | |||
| + | |||
| + | '''Hepatitis B inside modification''' | ||
| + | |||
| + | ---- | ||
| + | |||
| + | * 2nd try colony PCR of the transformations from 4. September | ||
| + | -> one colony seems to have the correct insert in the pSB1C3 backbone | ||
| + | -> no conclusion can be made about samples containing the Hep-inside coil fragment in the Bba_J04500 brick | ||
| + | |||
| + | '''GFP modification''' | ||
| + | |||
| + | ---- | ||
| + | |||
| + | * PCR of the transformations from 6. September (PCR step 1 in pJET) | ||
Revision as of 09:56, 18 September 2012
Hepatitis B general
- 2nd try colony PCR of the transformations from 4. September
-> one colony seems to have the correct insert in the pSB1C3 backbone -> no conclusion can be made about samples containing the Hep-inside coil fragment in the Bba_J04500 brick
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.
Hepatitis B inside modification
- 2nd try colony PCR of the transformations from 4. September
-> one colony seems to have the correct insert in the pSB1C3 backbone -> no conclusion can be made about samples containing the Hep-inside coil fragment in the Bba_J04500 brick
GFP modification
- PCR of the transformations from 6. September (PCR step 1 in pJET)
