How to conduct DNA cloning if plasmid's restriciton site is un-unique - (Aug/24/2008 )
Hi, I encountered a problem in my recent bio school paper and was wondering if anybody could give me their insights (:
I was given a DNA insert with PstI ends and was supposed to insert it into a plasmid vector. However, the restriction map of the plasmid vector had 3 PstI restriction sites. It had unique restriction sites of XbaI BamHII ScaI, but the DNA insert was not cut by these restriction enzymes. What should I do then? It seems an impossible question to me, but the teacher doesn't seem to think so could it be that I missed out anything?
Apparently the PstI site is not in MCS. You can desgn PCR primer with XbaI and BamHI site incoroporated into the primers and amplify your insert. You can also blunt your insert and vector and then do a ligation, but you have to screen clones that have the right orientation of insert.
ScaI produces blunt ends.
Ok thanks! So I take it that there's no way to insert a DNA fragment with PstI sticky ends into a plasmid with 3 PstI sites unless I do something exta like blunting the fragment's ends?
Is it an absolute rule that the plasmid must be cut only ONCE, at a unique restriction site? I need to be very sure of this before I go question my teacher xD
Yes. Or do as pcrman suggested and PCR amplify the insert and adding your desired restriction site. This method is the easiest, most versatile and often fastest.
It is possible (and people have done it before), to use a partial digest. Assuming one of the PstI sites is within the desired location which you want to insert your insert. You run a PstI digest, but only allowing it to go partway, so you have lots of single cut vector DNA.
You have to do a preliminary experiment first, to determine how much enzyme of PstI to add to X quantity of DNA, after leaving for Y time, to get single cut vector DNA.
You then gel purify the single cut vector, removing any double cut, triple cut vector. Once you have pure single cut vector you then ligate it to the insert.
Since you have three PstI sites, there are three positions which the PstI enzyme can make the initial first cut. And thus three places that the insert can go into. So the next step, you will have to screen for insertion of the insert into the right place. This scene can be done by PCR, or by restriction digest.
As you can imagine this method is not done often.
Another alternative is to inspect your vector for unique restriction sites of enzymes that produce cohesive ends that are compatible with PstI.
For example, NsiI (ATGCA/T) and PstI (CTGCA/G) produce compatible overhangs because of the TGCA common to their restriction recognition sites. Thus an insert cut with PstI could be "sticky-end" ligated into a vector cut with NsiI. Note, however, that the chimeric sequence produced by the ligation of these fragments will not result in either a PstI or an NsiI site (as it will be ATGCAG), so it might be a bit tough to recover your insert from the resulting plasmid, if that is required later on...
A table of compatible ends is available here.