getting rid of undigested vector - (Nov/08/2006 )
I am trying to clone a 1.8 kb insert cut with BamH1 into a Bgl II digested vector of 6.5 kb, and though it seems easy enough I cannot seem to do this. Upon doing all the controls I have found that the problem is that I have too many background colonies. When I transform DH5alpha cells with digested but unligated vector I get many colonies. I have tried to increase the amount of enzyme to 30U/2.5uL of miniprep DNA in 50uL total volume for one hour, still I have many colonies. I don't want to overdo it with the enzyme because I don't want to mess up the ends of my vector.
I am wondering does anyone know what I am doing wrong? When I gel purify my vector, it appears to be cut.
I wonder if there is a way to just stop fretting about getting a perfectly cut vector and moving on to the cloning. Would it be feasible to do the BamH1/BglII cloning and then add BglII to the ligation. The hybrid BamH1/BglII I have heard should not be recognized by either enzyme. If so, then how much RE should add? And should I add the RE during the ligation or afterwards?
Thanks so much!
One approach is to make the vector backbone with PCR rather than by cutting a plasmid. Make primers to the region around the MCS, with whatever cut sites you need, PCR around the entire plasmid, cut with the enzymes you are using, and go. Zero background. Custom MCS.
Use less vector DNA (~2ug) in a 30 to 50ul RE rxn. Gel isolate the cut fragment and avoid uncut vector.
Since you're using around 100 to 200 ng vector for each ligation rxn, using less vector DNA for RE is more than enough and it ensures complete digestion. 5U RE per ug DNA applies though.
what is happening is that your cut plasmid is pulling down some uncut plasmid DNA. This happens all the time.
What you can do is : -
1- Do two consecuitive gel purifications on your vector. After the first gel purification, place the gel slab in a new ge slabl, and run it again. This will help remove any pulled down uncut plasmid DNA
2. After the ligation, digest your DNA with BglII as you have suggested. However I would advice against adding BglII into the T4 ligase buffer, as the T4 ligase buffer doesn't have enough salt to support BglII's activity.
3. Live with it. And use colony PCR + a multichannel pipette in 10ul PCR reaction mix and test 96 or even 192 colonies. Brute force and ignorance...even in molecular biology still rules the day.
Have you used the dephosphorylated digested vector in ligation?
BamHI and BglII are compatible ends. So definitely dephosphorylate your vector as the vector itself can religate. I would also suggest to digest 2h that will really help as 1pg of undigested vector will be very efficient for transforming.