Hi Rohit, nothing unusual, cloning large fragments in large vectors with REs is always very difficult. Increasing the amounts of the DNA helps (for example up to 200 ng of vector, which will require at least 700 ng of insert), but it's not trivial since you have to gel purify the insert after restriction and the loss is high with large DNA fragments.

My suggestion is to go with Gibson Assembly (NEB) or In-Fusion (Clontech). You just need to design primers with a 5' tail that overlaps for about 15 nt with your vector, the rest is pretty simple and straightforward. A controlled 3'-5' exonuclease reaction will produce two complementary single strands on the vector and the insert and since the complementarity is 15 nt the annealing is very efficient. There are two good points that really increase efficiency: you don't need to restrict digest your insert, which means you just desalt it and you have higher amount availability. You don't have to ligate, due to the long overlap region, which is always the limiting factor in classical cloning. You only open up your vector (either with one RE or two, as you prefer,just design accordingly the primers for amplifying the insert with the correct homology region), then just put together vector, insert and enzyme mix for some time (see protocol) and finally transform.

Good luck!

Are your primers phosphorylated?

Hi Rohit, nothing unusual, cloning large fragments in large vectors with REs is always very difficult. Increasing the amounts of the DNA helps (for example up to 200 ng of vector, which will require at least 700 ng of insert), but it's not trivial since you have to gel purify the insert after restriction and the loss is high with large DNA fragments.

My suggestion is to go with Gibson Assembly (NEB) or In-Fusion (Clontech). You just need to design primers with a 5' tail that overlaps for about 15 nt with your vector, the rest is pretty simple and straightforward. A controlled 3'-5' exonuclease reaction will produce two complementary single strands on the vector and the insert and since the complementarity is 15 nt the annealing is very efficient. There are two good points that really increase efficiency: you don't need to restrict digest your insert, which means you just desalt it and you have higher amount availability. You don't have to ligate, due to the long overlap region, which is always the limiting factor in classical cloning. You only open up your vector (either with one RE or two, as you prefer,just design accordingly the primers for amplifying the insert with the correct homology region), then just put together vector, insert and enzyme mix for some time (see protocol) and finally transform.

Good luck!

Hello Stoyanov,

yes my primers are properly 5' phosphorylated.

Like what Pietro said, try doing Gibson Assembly, it can ligated in single reaction large fragments and very straight forward to. I did not have any problems with this system just make sure you have right overhangs, right components and competent bacterial cells. here is the link to learn more:

https://www.neb.com/products/e2611-gibson-assembly-master-mix

I've had to transform DH5a cells with large constructs before (17kb total size) and it is not an efficient process. So, this is what I do WHEN my constructs are larger than usual (assuming your insert and vector are actually able to bind):

For ligation: Ligate overnight at 16 degrees.The vector:Insert ratios you are using seem to be pretty normal, however, I have tried 1:15 ratio in the past and has worked for me.

For transformation: Transform your competent with your ligation mixture at a maximum 10:1 (cell:ligation) volume ratio.  For me, I transform 50 or 100 uL aliquots of cells with either 5 or 10 uL of my ligation mixture. Add a large amount of LB (at least 700 uL) and incubate at 37 for 2-3 hours (as opposed to 1 hour), then plate.

I have not tried Gibson Assembly before, but it seems like a good thing to try as well.

Good luck.

Thanks a lot Fabian, I will try it this week... !!! 

I would suggest you try cloning with In-Fusion (Clontech) method, but see that you have correct 15base overhangs you can do this in snap gene software. Once you have the correct primers with 15base over hangs you can perform In-Fusion method.

Instead of non-directional cloning perhaps you could try blunt cloning directly out of the PCR mix. Purify the PCR product, blunt it with an excess of dNTPs and some klenow fragment (or a dedicated blunting enzyme) and ligate into a blunt vector as Fabian has mentioned. Then once in this blunt shuttle vector isolate a large amount and do a very clean digestion with your two restriction enzymes and gel purify before trying to do directional cloning.

I recently cloned a few very AT rich ~6kb genes into a 7.6kb vector this way and it worked well. In our lab we use pJET1.2 blunt vector for a shuttle. We do all the sequencing while it is in pJET1.2 and only after transfer it to our expression vectors. pJET1.2 has the nice advantage of having a kill gene if it is empty when it closes, so it makes screening much faster. 

Best of luck with everything. 

Kristin, I still haven't succeeded. I only get vector dimers

Hi Rohit, Kristin was very thorough and very accurate in her last post, which is a very nice thing to see in today's superficial and fast moving world, and given your last comment I would suggest you to focus on two of her points in particular, if you want to stay with RE-based cloning (I still think In-Fusion or Gibson Assembly would make your life so much simpler...!). So, you say that you obtain only vector dimers, which when troubleshooting means two things:

1. You don't seem to dephosphorylate your vector (or not efficiently enough... otherwise it could not ligate on itself), so take care to have a good CIP treatment (I can suggest you NEB's Antarctic phosphatase);

2. You probably have a problem digesting your PCR fragment (otherwise with a ratio of 1:10 you would get your clones and not vector dimers), which, assuming that you are purifying your PCR fragment before digestion with an ethanol precipitation or a spin column, is nevertheless not unusual, since RE often do not work efficiently when their recognition sequence is on a DNA fragment end (whatever the NEB charts say, but you will indeed notice this much less on a small fragment...). To solve this problem I think the quickest way would be to subclone your insert into a TA vector (pCRII from Invitrogen or pGem-T Easy from Promega: all Taq polymerases, even proofreading, except Pfu, will add on a sufficient proportion of fragments a 3' terminal A that allows an efficient and quick TA protocol) and then cut it out of the TA vector with your enzymes that seem to work well enough on your vector and gel purify it. Alternatively you could design new primers with minimum 6 extra nt at the 5' end of the restriction site. Finally you might also think of using the KpnI isoschizomer Asp718I (that you can buy from Roche), in my hands has always worked much better than KpnI (BamHI should not be your problem, normally digests and ri-ligates efficiently in all situations).

Good luck!

Kristin, thanks for the suggestion. At present, I am trying to clone the amplicon in Topo-TA-XL vector (Invitrogen) and then proceed.