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about flanking sequnce


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#1 roshanbernard

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Posted 12 April 2009 - 04:57 PM

i am producing the synthetic gene using the Sal1 and EcoRV as the restriction site on 5' and 3' respectively...but the gene sige is only 36bp..and how do i increase its size just to use it appropriately in PCR and to get proper action of restriction enzymes...wat sequnce should i insert on 5' and 3'end...

#2 T C

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Posted 12 April 2009 - 07:13 PM

Hey,

I am not sure I get yr problem but if you are trying to ask about the number of bases that you need to keep on either side for the enzyme to act, then goto the NEB site and it will tell you. I generally choose the bases on the basis of sequence of the gene, if it is GC rich then A or T or vice versa, just make sure that you don't engineer a stop codon. :D

Best,
TC

i am producing the synthetic gene using the Sal1 and EcoRV as the restriction site on 5' and 3' respectively...but the gene sige is only 36bp..and how do i increase its size just to use it appropriately in PCR and to get proper action of restriction enzymes...wat sequnce should i insert on 5' and 3'end...



#3 laoz

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Posted 14 April 2009 - 07:40 PM

i am producing the synthetic gene using the Sal1 and EcoRV as the restriction site on 5' and 3' respectively...but the gene sige is only 36bp..and how do i increase its size just to use it appropriately in PCR and to get proper action of restriction enzymes...wat sequnce should i insert on 5' and 3'end...


I think you just ask company to synthesize two DNA oligos for you:
5' TCGAC...36nt...GAT 3'
5' ATC...complemental reverse 36nt...G 3'

let them anneal, you can ligate to where you want it insert.

Laoz

#4 roshanbernard

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Posted 14 April 2009 - 08:13 PM

thanks a lot laouz..i think i will do exactly wat u said..i will just give them a sequence and add randomly 6 bp on either end and ask the company to produse the gene and give it to me..




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