3 replies to this topic

[yusii]

Hi

im going to amplify a dna fragment, and i am planning to clone it into a particular plasmid/vector, and i can only do so by using only one ristriction site/enzyme as all the rest cut through my insert (pcr -product). my question is, how would i go about designing primers for my insert so i can use only one ristrection site?
any ideas would be much appreciated ! thanks alot

oh extra information - im using Gibson assembly to stick all my pcr products together.

Edited by yusii, 16 March 2013 - 08:13 AM.

[bob1]

Basically all you need to do is make the primer as you would normally (i.e. 18-25 bp, targeting specific sequences), but add a few basepairs 5' of this (usually 3 or 6bp) and then the restriction site, and finally a few more bp 5' again (this is to allow the RE something to bind to, most will not bind efficiently to a site at the end of a DNA strand). These additions always go on the 5' end!

You can check out the efficiency of the cutting near DNA ends in the Roche and/or NEB catalogues.

Your primer would look something like this for both primers:
3-6bp-REsite-3-6bp-specific primer.

[yusii]

Basically all you need to do is make the primer as you would normally (i.e. 18-25 bp, targeting specific sequences), but add a few basepairs 5' of this (usually 3 or 6bp) and then the restriction site, and finally a few more bp 5' again (this is to allow the RE something to bind to, most will not bind efficiently to a site at the end of a DNA strand). These additions always go on the 5' end!

You can check out the efficiency of the cutting near DNA ends in the Roche and/or NEB catalogues.

Your primer would look something like this for both primers:
3-6bp-REsite-3-6bp-specific primer.

thanks for the reply bob1, i have few questions though !
would the direction be a problem here, i mean the fragment can ligate either way? also what are these extra base pairs for " add a few basepairs 5' of this (usually 3 or 6bp) and then the restriction site"?
sorry for being slow !

[2xzwei]

If you cut with just one enzyme leaving sticky ends the insert could ligate both ways - wrong and right orientated - you can check that later on by restriction digest or sequencing your ligation product.

Extra base pairs are needed in order for proper binding of the restriction enzyme, it needs some space to "cling" to your DNA and start working. If your DNA insert codes for a protein to be expressed you should add bases corresponding with the Kozak sequence in front of the ATG start codon (e.g. GCCACCATG...).