7 replies to this topic

[Montys]

Hi everybody,

I should start with a new project in a new field. I should analyze an Protein. Therefore we want to look of different truncated versions of this protein (if it interacts with other, can fold properly aso.)

Now I have to make Constructs/Plasmids for each truncated version we want to test.

Questions are how to achieve this in the best way. I know that I can use specific primer, that map the region of the wanted truncated protein or full length protein, but how important is the 3'UTR region. The polyA I can get from plasmids, but should I if I take out a specific domain in the end of the gene/protein as in (Construct B) add the 3'UTR.

Example:
Construct A: 1---------2------------3----------4-------------5--------UTR----polyA
Construct B: 1---------2------------3----------4-------------
Construct C: 1-------------------5--------UTR----polyA

Do you have a good idea how to obtain the Construct C easily. I have to cut out right in the middle of the gene several domains. Of course you can try using enzymes but I am not sure if I can that way cut out ONLY the specific domains.

[bob1]

For expression from a plasmid the UTRs don't seem to be important in my experience.

[Montys]

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?

Edited by Montys, 11 July 2012 - 03:48 AM.

[bob1]

The usual ways to get the truncated forms are to either digest the full length (you might end up with some of the 4th domain in there though) or to PCR the exons separately and then ligate into a vector one at a time.

NEB's rebase says that cdp1 is not commercially available.

[Montys]

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.

[bob1]

You can do TA cloning with the A overhang Taq leaves.  Either way should work.  I would usually make primers with the RE sites on them..

[Montys]

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.

Edited by Montys, 27 July 2012 - 12:39 AM.

[bob1]

In theory any distance (so long as it is in phase) will work fine.  Longer linkers may interfere with the the protein conformation and solubility.