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Team:Groningen/12 July 2010
From 2010.igem.org
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These ligations were performed with the backbone that still contains the unwanted IS element with the PstI site. At the same time we are attempting to delete this element using the related plasmid pNZ8048. This plasmid is the same as pNZ8901 except that it has a nisin inducible promoter. A restriction of the two plasmids with XhoI and MunI and combining two of their fragments would produce the pNZ8901-bbs plamid without the IS element. | These ligations were performed with the backbone that still contains the unwanted IS element with the PstI site. At the same time we are attempting to delete this element using the related plasmid pNZ8048. This plasmid is the same as pNZ8901 except that it has a nisin inducible promoter. A restriction of the two plasmids with XhoI and MunI and combining two of their fragments would produce the pNZ8901-bbs plamid without the IS element. | ||
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[[Image:16-07-10gn.jpg|200px|thumb|left|left, pNZ8901-bbs. right, pNZ8048]] | [[Image:16-07-10gn.jpg|200px|thumb|left|left, pNZ8901-bbs. right, pNZ8048]] | ||
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| + | In both cases the plasmid was only partially digested. The pNZ8901-bbs restriction did give an excisable fragment. | ||
| + | The pNZ8084 restriction was repeated. | ||
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| + | [[Image:16-07-10-2gn.jpg|200px|thumb|left|]] | ||
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| + | Again, for some reason, the plasmid was only partially digested but an excisable fragment is present | ||
Revision as of 19:47, 23 October 2010
Hydrophobofilm
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A mix of the ChpE and ChpH genes were ligated into the pNZ8901-bbs backbone and a restriction check was performed on 8 clones. ChpE contains a DpnI site whereas ChpH does not. A restriction with DpnI and XhoI should yield a 487bp and 176bp fragment for ChpE and just a 648bp fragment for ChpH (+ a ~3500bp backbone fragment for both).
- 10ul plasmid - 1.5ul buffer R - 0.5ul DpnI - 0.5ul XhoI - 2.5ul MQ
Clones 2, 4, 6, and 7 contain ChpE, and 1, 3, 5, and 8 contain ChpH. Clones 1, 2, 3, and 6 were sent for sequencing.
These ligations were performed with the backbone that still contains the unwanted IS element with the PstI site. At the same time we are attempting to delete this element using the related plasmid pNZ8048. This plasmid is the same as pNZ8901 except that it has a nisin inducible promoter. A restriction of the two plasmids with XhoI and MunI and combining two of their fragments would produce the pNZ8901-bbs plamid without the IS element.
In both cases the plasmid was only partially digested. The pNZ8901-bbs restriction did give an excisable fragment.
The pNZ8084 restriction was repeated.
Again, for some reason, the plasmid was only partially digested but an excisable fragment is present
