Victoria- I'll cloned many a large fragment into lentiviral plasmids using Stbl3 and can assure you that EndA activity is not causing degradation of your insert. Most commercial DNA purification kits contain an optional wash step that allows you to remove EndA from your DNA. If you did have endonuclease A contamination, you'd see smearing of your DNA on the gel, and faint bands, which is evidently not the case for the gel image you posted.

Please provide some more details on your vector of interest, the cloning sites used, and the information for what conditions were loaded on to each lane of your gel.

Although Stbl3s are recommended for lentiviral constructs, we've had excellent success using JM109 which are EndA negative. I would highly recommend them over Stbl3 (better plasmid yields and purity).

One other cautionary note: lentivirus has a packaging limit for how much DNA can physically fit into a virion particle. Usually it is 10kb. I've successfully gotten 12-13kb constructs to work, although titers are quite low. I highly doubt a 16kb insert could fit, especially considering the cPPT, LTRs, and other viral elements account for several additional kilobases of DNA.

Victoria- I'll cloned many a large fragment into lentiviral plasmids using Stbl3 and can assure you that EndA activity is not causing degradation of your insert. Most commercial DNA purification kits contain an optional wash step that allows you to remove EndA from your DNA. If you did have endonuclease A contamination, you'd see smearing of your DNA on the gel, and faint bands, which is evidently not the case for the gel image you posted.

Please provide some more details on your vector of interest, the cloning sites used, and the information for what conditions were loaded on to each lane of your gel.

Although Stbl3s are recommended for lentiviral constructs, we've had excellent success using JM109 which are EndA negative. I would highly recommend them over Stbl3 (better plasmid yields and purity).

One other cautionary note: lentivirus has a packaging limit for how much DNA can physically fit into a virion particle. Usually it is 10kb. I've successfully gotten 12-13kb constructs to work, although titers are quite low. I highly doubt a 16kb insert could fit, especially considering the cPPT, LTRs, and other viral elements account for several additional kilobases of DNA.

Like Daniel said, most kits have a wash step to get rid of EndA during plasmid purification. Moreover, that gel does not look like DNA affected by EndA. I had a problem with EndA once, and I got one big smear on my gel and not unexpected bands.

Hi Daniel and Ilya. Many thanks for your quick replies. It seems that EndA activity is unlikely to be part of my problem - and indeed reading more about my mini-prep kit, there would appear to be steps to eliminate this as an issue. I will try cloning my fragment into a slightly smaller vector we already have in the lab, which will result in a 14.5kb construct. May still be pushing it from what you say Daniel, so I will also investigate other smaller transfer vectors in the meantime - as the biggest insert I am trying to clone in is 4.4kb. Many thanks for your replies.

Dear Victoria, the other problem that you may facing is that you have, probably, not too pure insert and you may have different clones from different bands. I advice to clone by converting your vector into a T-vector, that is very easy. To avoid any background, you should digest the vector and extract the band from the gel. Follow this protocol: http://www.uvm.edu/~tpdelane/lab/protocols/Tvector.html

It is specially useful for big vectors and big insert. Good luck!!

Hi Alexandro, thanks very much for your reply. However I have subcloned my fragment by an EcoRI digest from the parent vector then blunt-ended it in order that it should be compatible with my BamHI (and then blunt-ended) vector. So, presumably creating a T-vector from this blunt-ended vector and then treating the insert with dATP and Taq to create an A overhang might increase ligation efficiency due to the artificially created 'sticky end'? Do you think this is feasible/worth doing?

Victoria- I suspect the problem with your ligation is the fact that you are using blunt ends. Blunt end ligations have much higher vector background rate (i.e. recovery of parental plasmid) and are especially challenging for large inserts, in my experience. I would suggest that you try recombination mediated cloning (Gibson method or Gateway technology) OR use PCR to change the restriction enzymes available for cloning (if possible, change to two different enzymes at the 5' vs 3' end to control the orientation of the insert). If you decide to stick with the blunt-end ligation, you'll probably need to CIP treat your vector and perform the ligation at 14C overnight.

Yes, Victoria. This procedure will increase efficiency by two order of magnitude. The best procedure should be amplified your insert by PCR with taq polymerase, while you digest and treat your vector. Then you don't need to treat the insert with dATP and Taq to create an A overhang. If you do that, and use a donor vector for PCR template, treat the final PCR reaction with DpnI to eliminate the template DNA, then purified the PCR product using an standard clean-up kit and then, perform the ligation. Since ligation of vector-vector and insert-insert is almost null in this procedure, you shouldn't have almost background. It is feasible and non labor-intensive, even, you can do it in parallel with your current strategy, always use fresh PCR product and be careful with vector that is prone to loose the overhang if diluted in water. The best is elute it in Elution buffer from the kit, it is Tris-HCl 10 mM, which do not affect any subsequent reaction. My other advise is to use no more than 50 ng of DNA for your standard restriction enzymes-based method, something that is going to diminish the background. If after ligation proceed to inactivate T4 ligase for increasing the transformation efficiency.

Thanks for the further information Alexandro. I will order some primers to PCR out the fragment from the parent vector. However in the meantime whilst waiting for them to arrive - I may try adding the A base onto my already prepared blunt ended insert - do you think this is worth giving a try? Theoretically I can't see why not, but am far from an expert on these kind of methods. Many thanks again.

Taq polymerase could introduce mutations in such a large insert. Although it can be used, it is risky for targets of 3kb or larger.

Hi Daniel. Yes, very good point - but im not PCRing my fragment - ive already subcloned it, im just planning to use the Taq to put on an extra A base on the 3' end, which apparently is theoretically possible according to this paper http://www.ncbi.nlm.nih.gov/pmc/articles/PMC333800/ cited in the method Alexandro posted earlier.

Yes Victoria. You can do it with your fragment, it work perfectly. Follow this simple protocol from Promega: http://promega.wordpress.com/2010/01/19/a-quick-method-for-a-tailing-pcr-products/

At this point you have nothing to worry about mutations because you are not going to amplify anything with your fragment (you have no primers!), only to take the advantage of this taq property to add A overhangs.

Regarding to PCR product with large insert, it is not so risky as people thinks it is. This is fair enough for companies that what to sell more expensive polymerases. I work in the field of mutagenesis in bacteria and the probability to fish exactly a mutated clone in a fragment of 5 Kb is 1 in 100. So, normally you preserve at least free positive clones and, at least me, always perform a quality control by sequencing. Nowadays is very cheap and fast. Nothing to fear by using tag. Cheers.

If ligation is not good enough, or DNA fragments are damaged badly by UV when gel extraction, you would get more background colonies which have truncated, selfligated plasmids etc. It happens even if you use EndA-

Thanks again for your replies. I have just Klenow treated my new vector and put it through a column to get rid of the RE, Klenow and importantly dNTPs. Im planning to do the Taq treatment of both this and my inserts in the morning - normally I would have dephosphorylated the vector at this stage with Antarctic Phosphatase - but have not done so due to the Taq step - which should reduce vector alone religation to pretty much zero if it works efficiently? Many thanks for the continued advice - I hope this works!

And also, a more basic commentary than our peers' which are very precise on their suggestions... Maybe you could load less material on your gels. They seem a little overloaded and that can also cause apparent smearing. I hope by now you already have your clone!