list of sequence to be added before RE site - (Sep/15/2009 )

hello, i would like to construct a primer for the first time that contain a RE site for ligation next time.

I want to ask where i can find a useful link to find additional nucleotide sequence that will flank my RE site. Thank you.

I guess you want a primer that will add a restriction site to both ends of your gene of interest?

If so, I recommend the following recipe.

5' :
Six random bases - your restriction site- quite a few nucleotides of your gene of interest's sequence 5' sequence, including the ATG.

3' :
Six random bases - your restriction site - quite a few nucleotides of your gene of interest's sequence 5' sequence, including the stop codon.


Here's an example of what I did once. I had to add restriction sites at both ends of my sequence. It gave the following result.

5' GGT AGG CTC GAG ATG GCC TGC CTC CAT GAA ACC
Random XhoI GOI 5' sequence with ATG

3' GAG TAA GCGGCCGC TCA TGG AGG AAG GGA GGC
Random NotI Inverted 3' sequence with stop codon



And make sure the restriction sites you add are not found in your sequence

I found this site useful:

http://www.neb.com/nebecomm/tech_reference...nucleotides.asp

Take note of their cleaveage percentage for 2h and 20h...if enzyme can digest for >2 hrs, I usually only look at 20 h...

Hope it helps...

thank you so much guys...