urgent HincII digestion problem - (Apr/14/2007 )
Please can you help? I am trying to cut an 8.6kb non-commercial plasmid (i.e. has no blue/white selection) with HincII and BamHI to insert a 50bp insert but I have problems! When I do a single digest of the plasmid with BamHI but when I do a single digest with HincII it refuses to cut.
I am aware that HincII is succeptible to CpG methylation but I have checked my plasmid and this has not happened. I have also tried getting enzyme from different suppliers but this also hasn't worked. Using SalI (which cuts at the same site but giving overhang ends) I get a 50% digestion from an overnight incubation so the site definitely exists.
Moreover when I do a double digest with HincII and BamHI, I appear to be able to reduce the size of the linear fragment by about 900bp which is the correct size (although this 900bp fragment does not appear on my gel when I purify the linear band) however I do get a lot of smearing on the gel (too much protein??).
I was thinking that maybe EDTA might inhibit HincII but am not sure, also if HincII prefers a linear substrate but I have no proof of this as nobody else seems to have a problem with this enzyme. Please can anyone help?? The protocol gets more complicated further down the line and so to be stuck digesting my plasmid makes me kinda nervous!!
Hope to hear from you soon,
I don't understand what your final goal is. Why would you want to cut with a blunt ended cutter rather than the sticky ended cutter? Why not use SalI, since you know it works for you? (Admittedly, SalI has issues, but apparently not as many as HincII.) EDTA will certainly inhibit reactions, but think about how much is present -- the RE buffers typically will provide enough Mg++ to overcome the modest amounts of EDTA in TE dissolved DNA.
Thanks for your reply. I'm trying to cut the plasmid with HincII to give a blunt end and BamHI to give a sticky end to ensure that the insert goes in unidirectionally.
Unfortunately within the plasmid the HincII site is the only blunt end cutter available in the area (before starting to affect areas of RO and antibiotic resistance). By cutting it with SalI I was trying to insert another oligo with more unique blunt end restriction sites that I could then maxi-prep up to give me more flexibility.
Thanks for the info on the EDTA - it was something I really wasn't sure about and so it's good to know that in all probablity it's not going to influence my reactions.
Two different sticky ends will work much better for assuring that the insert goes in unidirectionally.
In using two different sticky ends would I still need to CIP treat the plasmid?
Thanks for your help,
No. This is one of the good reasons to work with double-cut vectors. While some people make them work well for them, I have never been able to make phosphatases play nice for me.