Restriciton digest protocol for adjacent sites? - (Jan/24/2008 )
Does any body have a protocol to do a double digest on adjacent sites that cannot cut when both enzyme are used at the same time?
I'm trying to clone a PCR product between them and i have no other choices.
many thanks in advance.
If your PCR product is not extremely long you could try the QuikChange strategy from Stratagene. This can be used to insert a PCR fragment independently of restriction sites. Read about the "site directed mutagenesis" on their web-page and then simpply use your PCR product (you may need to design new primers so that the product overlaps your destination-vector) as if it was two long "QuikChange-primers". This idea is found in the paper: "Integration of PCR Fragments at Any Specific Site within Cloning Vectors without the Use of Restriction Enzymes and DNA Ligase", BioTechniques 31:88-92 (July 2001). Good luck
do you have a pic or image of how the sites are located?
Could you digest them, sequentially?
You can pcr amplify the plasmid with primers which include sufficient extra bases 5' of each restriction site. PCR purify and then cut the linear fragment with your two enzymes.
My insert is large, ~3kb so the quick change method is out.
I'm using a pXJ40HA vector
----BamHI/HindIII/XhoI/NotI/SmaI----
I'm trying to cut HindIII and XhoI in one case, and in another, XhoI with NotI. these cut the vector when used one at a time.
but sequentially, the second enzyme doesn't cut, according to NEB.
The inserts were amplified with primers that include the sites and extra 4bp at the 5' ends of the primers.
If anyone has the full sequence of pXJ40HA please let me know too.
Many thanks again
I have a work around but it requires a bit of extra cloning. First, clone any XhoI fragment you have into the XhoI site (it doesn't matter what this is but preferably something fairly different in size to the vector). When you have this, simply cut the vector with either HindIII or NotI as appropriate. This will linearise the vector. Now clean this up and cut with XhoI (which will drop out the fragment you cloned in). Then the vector you have left will be cut with both enzymes. A bit of a pain to get the initial vector with insert but probably worth it if you have to use these sites in this vector a lot.
i use a 2 step protocol :
in tube 1 digest 1µg DNA with XhoI (1µ)
in tube 2 digest 1µg DNA with HindIII (1µl)
go for overnight digestion
the next morning mix tube 1 and 2, add 0.5µl of each enzyme and go for 3h.
Purify and go for ligation.
Even though the suggested methods are preferable, you can go way above 3kb with quickchange. (Go up to 8 kb myself regularly without problems, not using the kit btw).