restriction enzymes/sites to avoid (View forum version)



lyok

Posted 11 April 2014 - 09:51 AM

Dear all,

 

I can (vaguely) remember reading something about restriction enzymes that you should avoid when doing cloning.

Too bad, I forgot what exactly it was and I also cant remember the enzymes..

 

Anyone an idea what restriction enzymes/sites one should avoid for cloning?

 

 


jerryshelly1

Posted 11 April 2014 - 11:20 AM

If you are using a manufacturer provided plasmid, chances are each RE present in the multiple cloning site has been thoroughly tested and will give decent results. I tend to avoid RE sites that produce weird overhangs or require any (N) nucleotide to cut. Always avoid compatible end producing enzymes (isoschizomers)


phage434

Posted 11 April 2014 - 11:35 AM

SalI has a bad reputation when used as a site created by placing it on the 5' end of a PCR primer.

I always choose enzymes which can be heat killed, which often avoids a purification step.

Choose cheap enzymes.


lyok

Posted 12 April 2014 - 09:18 AM

SalII thats it!

 

I remember this. But why would it cause problems ?

 

I often dont even do a purification step.. Even heat inactivation is something I often dont do when transforming cells. So far I had no problems with this.

 

 

@jerryshelly,

 

what do you mean by "weird overhangs" ?

 

And require any nucleotide to cut? You mean the standard "rule" they often mention that you need some extra bps to have it cut your restriction site?

 

Isoschizomers, I can see this also as an advantage , but I guess this depends on the reason why you use it.


jerryshelly1

Posted 14 April 2014 - 01:06 PM

Weird overhangs such as AleI that produce weird cut sites with "any" nucleotide. I just try to avoid using these all together.

 

Ale-I-cutsite_1_v1_000007.gif

 

Edit - I guess they are not necessarily weird, I just prefer not to tinker with them. I like my common REs.