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Team:Wageningen UR/Journal/week20
From 2012.igem.org
week 20: 10 September - 16 September
Hepatitis B general
10 September
- 2nd try colony PCR of the transformations from 4. September (with sequencing primers)
-> 6 out of 9 samples show an insert of the right size
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
10 September
- 2nd try colony PCR of the transformations from 4. September (with sequencing primers)
-> 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
10 September
- colony PCR of the transformations from 6. September (PCR step 1 in pJET) - using pJET sequencing primers
-> no band on the gel
11 September
- step 1 of the PCR reaction
- With different template concentrations (from 20ng until 0.2ng added to the mix) - With two different primer sets (both with the forward primer adding part of the coil + once with the GFP reverse primer adding a histag at the C-terminal and once with the sequencing reverse primer) -> bands of the expected size visible (even the samples that contain a very low ammount of template). There is also a second band visible (decreasing in strenght with decreasing template concentration) -> something went wrong with the second set of reactions (the reactions where made in duplo)
-> this shows that the experiment can be continued with the sample containing only 0.2ng template DNA (so the risk of transforming with the original template is lowered)
- Of the GFP-coil 1st step (both primer sets) in pJET (samples with 0.2ng template in the PCR mixture)
- Transformation With DH5α using different amount of ligation mixture for the electro transformation (1, 3 and 5 µL)
-> more colonies where growing on the plates containing transformants transformed with 5µL
12 September
- Colony PCR of the transformations (GFP-coil step1 in pJET in DH5α)
-> the transformation was succesfull - there are colonies present that contain an insert with the expected size
