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Design primers for expression cloning

primer cloning expression start codon

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11 replies to this topic

#1 memeta

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Posted 14 March 2012 - 07:18 AM

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.??Posted Image Posted Image


hoping for ur answer
Posted Image

#2 bob1

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Posted 14 March 2012 - 01:42 PM

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.

#3 memeta

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Posted 15 March 2012 - 05:11 AM

thank you, that really useful.Posted Image Posted Image

#4 memeta

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Posted 15 March 2012 - 05:22 AM


(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

#5 bob1

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Posted 15 March 2012 - 12:12 PM

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.

#6 memeta

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Posted 20 March 2012 - 02:09 AM

thank you so much for answer

#7 memeta

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Posted 20 March 2012 - 02:51 AM

actually I have a problem in designing my primers I need an urgent helpPosted Image . 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 ???Posted Image

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 ?Posted Image
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?Posted Image
and thank u in advancePosted Image and sorry for the length of thisPosted Image

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


#8 phage434

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Posted 20 March 2012 - 05:27 AM

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.

#9 memeta

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Posted 20 March 2012 - 07:24 AM

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 highPosted Image as we were told Ta should be between (52-58) but I am not surePosted Image
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?? Posted Image

#10 pmggms2

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Posted 05 June 2013 - 12:36 PM

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.

Question so why do you add random bps at the beginning of the restriction enzyme

#11 bob1

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Posted 05 June 2013 - 12:51 PM

Question so why do you add random bps at the beginning of the restriction enzyme

So as to give the RE something to bind to - most of them won't work very well close to the end of a sequence, so you need a few bp (6 is usually enough, some will work with less) beyond the restriction site.

#12 pmggms2

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Posted 06 June 2013 - 06:42 AM

thanks





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