We need a cloning vector with Sfi I restriction site. We would be grateful if anyone can provide the same to us.
you can insert a cassette which contains Sfi I restriction site into your own cloning vector by digest two different restriction site of it.
For exemple : if your vector's MCS contain BamHI and EcoRI site,you can digest your vector by the above two enzymes,and then you can design a pair of oligos which Sfi I restriction site with the BamHI and EcoRI Sticky ends which like that :
FOWARD: GATCCAAGGCCNNNNNGGCCTTG;
REVERSE: AATTCAAGGCCNNNNNGGCCTTG
Annealing the above two oligos(Boiled the two oligos in 50nm NaCl at 100℃,and then natural cooling to room temperature) , they will become the pattern of the attachment .
you ligate this cassette to the your own vector which be digested by BamHI & EcoRI ,you will find you have got the cloning vector with Sfi I restriction site.
Thank you very much Mao. We will design the cassette with the Sfi I site and insert it in the vector which we have in our lab. This would be a better option as we can use the same vector that is suitable for our work.
pay attention to Sfi I：1、Methylation Sensitivity；2、SfiI requires two copies of its recognition sequence for cleavage to occur. The two sites can be on either the same or different DNA molecules.
Does this imply that two recognition sites should be incorporated in the plasmid or 1 will do, as the restriction reaction will have several DNA molecules? Or is it that it should be two different DNAs or plasmids?
Like this:
foward: gatccaaggccnnnnnggcctggtcggaggccnnnnnggccttg;
reverse: aattcaaggccnnnnnggcctccgaccaggccnnnnnggccttg
Isnt it that Sfi requires a 200bp stuffer in the region to act at that site ?
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