Do I have to purify vector after RE digestion for cloning? - (Jul/25/2006 )
i know i sound like a beginner 
...but i usually purify PCR insert (from PCR reaction stuff) and digested vector together from low melting temp gel, phenol chloroform etc....but today i got the purification kit for PCR products.. 
....so I am wondering now, if i make sure that the vector is completly digested should I purify it? can't I use it directly for cloning? i mean some invisible portion might not be completly digested but negative control should rule that out no? please please tell me i dont have to do purifications... 
i would definitely purify vector before ligation........it doesn't take that long and means you won't have to screen so many colonies!!!
I purify the vector because sometimes you cannot digest 100% (always I would say, even if you think it's 100%) and on the gel you can separate the digested band from the non digested one. That will allow you to screen less colonies as aussieuk said
ok
there are reliable methods for vector purifications and they depends of the size of the fragment removed by restriction digestion.
When i digest a polylinker site, i purify it on column. For fragments up to 400bp, columns are a good and fast way to purify.
For more than 400bp, you need to purify on gel (method which is not my favourite, as you may have noticed in my preious posts )
Do a negative control should give you an info of the relative amount of undigested.
But for sure a purification step is necessary to reduce this background.
You may consider also the fact that, even if you gel purify your vector, there is undigested or simple digested remaining.
You can eliminate background almost entirely by preparing your vector backbone by PCR. Since there is very little template circular vector present, the only replicating vector is the result of the ligations.
thank you everyone, phage, please can you elaborate more on the idea....i think i dont quiet got it...
Make two primers which prime outward from the multiple cloning site. Keep as much or as little of the MCS as you like, and insert whatever restriction sites you want in the 5' end of the primers (remember to add 6 bp of 5' junk sequence to be cut off). PCR small amounts of DNA containing your desired vector backbone with these primers. PCR cleanup, cut with the enzymes, purify if you want (we use Microcon filtration units to remove the short dsDNA fragments). Ligate and go. You never need to worry again about uncut plasmid transforming, and you have complete flexibility in your choice of cloning sites.
I also purify my vector in a gel to eliminate possible background and I can tell I always get 100% when doing colony PCR after transformation I've even screen about 80% of my colonies still get no background.
Kathy don't avoid away from the gel purification after cleaning with PCR purification kit.
Kathy don't avoid away from the gel purification after cleaning with PCR purification kit.
I prefer to gel purify to avoid any background colonies. I know its not my favorite procedure but if things work with it, get used to it.
I also purify my vector in a gel to eliminate possible background and I can tell I always get 100% when doing colony PCR after transformation I've even screen about 80% of my colonies still get no background.
Kathy don't avoid away from the gel purification after cleaning with PCR purification kit.
I prefer to gel purify to avoid any background colonies. I know its not my favorite procedure but if things work with it, get used to it.
I understand that purify prevents background...but what if your problem is that you can't even get the vector to religate as a positive control.
Thanks ahead