How about vector sequence in cloning? - (Feb/16/2008 )

As a beginner in Molecular biology, I have a question about cloning.
say, the MCS as showing: KpnI----NcoI----NdeI---SacI. Now I cloned my gene into KpnI---NcoI and cloned a tag, such as GFP into NdeI----SacI. Now my question is:


1, Is the sequence between NcoI---NdeI translated into amino acids? if yes, Does this peptide affect the function of my gene products?
2, Does the gene have to delete the stop codon since I have a tag at N-terminus?


Thanks,


triticum

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You have sort of answered your own question here. If you have a stop codon in your insert, the NcoI to NdeI won't be translated. If you don't have anything to stop translation, you will keep adding amino acids until your transcript reaches a stop codon. Unless you have a fairly long tail of these extra residues, they sholdn't affect your gene product too much, if at all. Having said that, they might, the only way to know for sure is to do the experiment and see what happens.

Good luck with it.

swanny

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if you have the gene 5' in KpnI, and tag between Nde & sacI, then the tag is in C-terminus.

And if you clone a tag before the 5'of gene, then have the tag starting with start codon and let it continue with the gene and end as it would at the stop codon.

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I appreciated the two answers and that make me more clear.

But I still have a bit confusion about with or without stop codon.


Actually there is mistake in my second question. the tag should be at the C-terminus, not at N-terminus.

OK ,now:



Promoter----KpnI-----(my gene)----NcoI---NdeI---(GFP)---SacI.

as shown above, In order to localize my gene products I use a tag (GFP) at the C-terminus, now my questions are:

1, Should I delete the stop codon of my gene? if my gene with a stop codon, can GFP localization still reflect the real location?


Thanks a lot,

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Definitetly you have to eliminate the stop codon from the end of your gene. And be careful to clone GFP in frame with your gene! So you will get a GFP fusion protein product.
att

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Yes you must remove the stop codon from your favourite gene (yfg), else the GFP will not be translated. What you want is a yfg-GFP fusion gene, which transcribes to a fusion mRNA. As there are bp separating the two genes, you will have to make sure that the GFP is still inframe with the yfg. Also inclusion of a fexible loop should be considered, a spacer to keep the two proteins from getting to close to each other that they may intefere with each other.

I am uncertain why do you mean by 'real' location.

• If you mean 'rea'l location as the GFP indicating which cells that have taken up your fusion gene construct then most surely.
• If you mean 'real' location as the GFP indicating where yfg is within the cell.... well maybe... it is not gurenteed. Fusion proteins occasionally have altered properties.

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Thank you guys so much! it has cleared most of my confusions.

But still,

1, What is the flexible loop; is it just a any long spacer or is there any special requirement for that spacer? Do you have any paper mentioned for that?
2, if my construct is like : yfg with stop codon------ ATG-gfp(means with its own ATG). So can gfp be translated?


Thanks a million!

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I don't have anything with me at the moment.

No, it won't. The ribosome will primarily just drop off.

If you want separate yfp and GFP proteins from a single mRNA transcript... you may either consider either adding a self cleaving peptide or an internal ribosomal entry site. By separating the yfg from the GFP you won't be able to say of if the GFP is colocalising with the yfg. Only that this particular cell has your gene construct.

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If you do not need to have GFP fused with yfg it might be easier for you to use GFP construct that has a MCS with GFP under a separate promoter. I'm currently using pTRACER vector from Invitrogen. You can use the pTRACER vector that has a Anti-V5 tag sequence after the MCS and thereby allowing you to fuse the tag to yfg (make sure you remove the gene's stop codon and that it is in frame). Then you can use GFP status to indicate cells that have your plasmid and then use an antibody to the tag to find out where yfg is localizing in the cell. I've done this - you can see a pic in another forum topic. I've included the link below.

http://www.protocol-online.org/forums/inde...c=34438&hl=

In my case the GFP localizes to the cytoplasm but my gene localizes to the nucleus.

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Thank you guys very much! it helps a lot!

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