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  2. → Viewing Profile: Posts: Montys

Montys

Member Since 15 Apr 2001
Offline Last Active Aug 11 2012 08:41 AM
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Posts I've Made

In Topic: How getting truncated DNA fragments?

27 July 2012 - 12:36 AM

Hi,

I have another question regarding this nice project. Do you have an idea what the maximum distance between my specific protein and my epitope tag could be. I have the problem that I can not ligate the gene directly with the epitope sequence. So there are minimum 12 bases in between. I often don't see any information in other papers. One showed 6 bases.

Example for C-terminal tagging:

Gene-xxxxxxxxxxx-Tag

Would be great if somebody has an idea.

In Topic: How getting truncated DNA fragments?

11 July 2012 - 10:48 PM

Damn it! Yeah as I thought. So a lot of work to do.

I will try to PCR the Exons. Hopefully I get good PCR results. I hate it when I get mutations because of lousy primer, due to the fixed positions the primer have to anneal.
Do you just make the PCRs or do you do  after the exon specific PCR an additional PCR for generating good sticky ends. The Taq it self makes already sticky ends, that should be enough or not?

Thx so fare. We will see what the next weeks will bring. The group I work together with thinks you can manage such thinks in 2 weeks. I calculate more likely 2-3 months. Hopefully the protein is in the end expressed and can be purified with the Flag-Tag.

In Topic: How getting truncated DNA fragments?

11 July 2012 - 03:00 AM

Thx. I also thought that way but wanted to make sure.

Du you have a nice idea how to get a domain in the middle of a gene truncated as mentioned in Construct C.

By the way does anybody know the endonuclease CdpI I couldn't find it. Either NEB nor Fermentas or Promega seemed to have this enzyme. With this Enzyme I could cut the last domain of the Gene. Strange in SerialCloner the enzyme is shown?

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