8 replies to this topic

[memeta]

Hi everybody,

I want your help

I am trying to design primers for amplification, cloning and expression of my gene. I want to insert the gene in pGEX aT1 vector which is a GST vector (for fusion tag) and i am wondering if i should exclude the start codon, the stop codon( have them in my gene)  or neither are correct.??


hoping for ur answer

[bob1]

It depends on if you are tagging N-terminally or C-terminally, or both.  In the case of an N-terminal tag, the GST will already have a start codon, but you can still leave the one found on your gene in, if you like, it will make a methionine in the aa sequence.  You can also cut it out if you like, it probably doesn't make much difference.  Many people would say to remove it to prevent skipping of the RNA polymerase.  You should include a stop codon for either of these situations. case!

For a C-terminal tag, you will need to include a start codon to make sure that the gene transcribes.  However, you must remove the stop codon to prevent the transcript terminating before the tag.

[memeta]

thank you, that really useful.

[memeta]

(It is important to remove start codon of the protein when tagging it on the N-terminus to avoid an expression of untagged form of the protein. You have to remember that promoter will drive expression of any open reading frame that is downstream of it. It is also crucial to remove stop codon when we tagging protein on the C-terminus to prevent premature termination and allow expression of a fusion protein). I read this in one of the website but I don't think it's true because my sequence contains many MET residues which could cause the same problem even if I  exclude the start codon, what do u think about ?this sentence

[bob1]

They are absolutely correct, however, the first open reading frame should be the one that is transcribed the most; the liklihood of skipping decreases as you get further from the promoter.  If you are using a short tag (e.g. V5) or have particular sequences in your gene that the promoter uses to enhance transcription, this could be a problem.

Find out if your GST tag has a kozak sequence - this should (in theory) minimise any chance of skipping.

[memeta]

thank you so much for answer

[memeta]

actually I have a problem in designing my primers I need an urgent help . I have this sequence

ATGGATGATTTGATGCTGTCCCCGGACGATATTGAACAATGGTTCACTGAAGACCCAGGTCCAGATGAAG-------------------------------------------------AGGGTCAGTCTACCTCCCGCCATAAAAAACTCATGTTCAAGACAGAAGGGCCTGACTCAGACTGA

1- reverse   primer ( 3 CCGGACTGAGTCTGACT+ GAATTC+ GCT 5) AT=54    
2- forward   primer ( 5 ATGGATGATTTGATGCTG + GGATCC+ TGA) AT= 50
What do u think in these primers ???

I chose these sequences as my  primers (18/17pb +6 for restriction site +3pb to increase the enzyme efficiency) but the problem is the annealing temp for the second one is just 50 an d we have been told that it should be between 52-58!!! what do u think ?  
Another thing is the primers length should it be 18-24 with or without the restriction site?? and should they have the same nucloetides number or it fine for 1 or 2 nucleotides difference??
and how many c or g should i have at  the 3 end as a CG clamp?
and thank u in advance  and sorry for the length of this

Edited by memeta, 20 March 2012 - 02:55 AM.

[phage434]

First, I would recommend always writing primers in the 5' to 3' direction.  Suppliers expect that order, and it is also very conventional in papers.  If you rewrite your reverse primer in that orientation, it is:
5' TCG GAATTC   TCA GTC TGA GTC AGG CC 3'
The binding portion of this primer seems a bit short (17 bp), but given the GGCC at the 3' end, I would probably leave it, especially since the next base would be C.  If you knew bases 3' of the end of the sequence you have given, I would add 2-3 just before the TCA in the primer above.

The forward primer is wrong.  The restriction sites are always at the 5' end of the primer.  You want something like this:
5' TGA GGATCC   ATG GAT GAT TTG ATG CTG 3'
Here, your short binding region really would be a problem.  I would recommend extending the primer by adding the next few bases:
5' TGA GGATCC   ATG GAT GAT TTG ATG CTG TCC 3'

Are you sure  you want to use BamHI as an enzyme?  It can't be heat killed, unlike most of the alternatives.

[memeta]

Thank you very much for ur answer.
ya we have been told to use this enzyme because it is available in our lab.
If i add few bases to the forward the Ta will increase to 74 , dont u think it's very high as we were told Ta should be between (52-58) but I am not sure
I can add some nucleotide to reverse primer and it/'s gonna be 5' TCG GAA TTC GTA TCA GTC TGA GTC AGG CC 3 but the problem is the stop codon I have my stop codon at the end of the sequence??