Blunt Cloning Large Insert/Smaller Vector - (Jul/23/2007 )
Hello everyone- I need some suggestions on how to proceed with a difficult cloning...I am trying to blunt clone a 9.9kb insert into a 6.1kb vector. I have very limited choices regarding RE sites, hence the blunt cloning. I have to cut this vector with AscI, and I cut my insert out of the current plasmid using NheI/MluI. After heat killing REs, I am using Klenow +dNTPS to fill in and create blunt ends, then heat kill both sets of reactions. I proceed to gel purify the insert using Qiagen kits, while I proceed to dephosphorylate my vector using Antarctic Phosphatase; heat kill the phosphatase, then column purify vector using Qiagen kit.
As might be expected, I am still getting plenty of self--ligation on vector control plate (trying now to up dephosphorylation time to 1 hour from 30 minutes) Ligation strategies tried to date: a) using regular T4 doing o/n ligation at 16 degrees, 1:3 and 1:5 vector:insert ratio using either 100 or 75 ng vector; 2) NEB Quick Ligase kit ( has PEG in buffer), same ratios; 3)Caculating ratios based on insert size as vector (in other words, calculating ratios in reverse) while still dephosphorylating vector; 4)Am proceeding to increase vector:insert ratio to 1:10. Doing PCR colony screen and /or restriction digests on plasmid miniprep DNA- will go to colony hybs if I have to.
Any novel ideas on how to cram a big honkin' piece of DNA into a smaller piece? By the way, don't think I can subclone in the insert in pieces-all my RE sites are also in the vector.....Any and all suggestions will be welcome...Many thanks, jack
well for this kind of nightmares, i would recommend you to by a duplexes of oligos in order to create novel MCS site. Aneale oligos are more effcient for ligation. Then you may ligate your insert in non-blunt-manner.
for blunt end dephosphorylations, promega procedure says : add 0.5µl phosphatase, 15' at 37, 15' at 56, re add 0.5µl phosphatase and re do 15' at 37, 15' at 56.
if you obtain so much self religation i would also increase digestion times for vector preparation.
Increase the time you digest your vector, and if possible: gel purify it to get rid of uncut vector. Creating a novel MCS might facilitate things as Fred suggested (you might do this by annealing oligo's or by "site directed mutagenesis" using primers containing restriction enyme recognition sites, I've gone beyond 6.1 kb with great succes).
Thanks, Varius and Fred 33...I will also try increasing digestion time and possibly gel purifying to reduce vector background....But it seems both of you are suggesting alternate strategy away from blunt cloning altogether...how much longer do you think I should try to pursue getting this through the blunt cloning method....Many thanks again, jack
I'm not an expert in cloning, so it might be stupid. But I learnt, that ligation used to be done at 4 degrees. Maybe the reaction would go much slower than at 16 degrees, but it may slow down your DNA a bit, and they'd be happier to stick together.
Hi Kupac-Thanks for the suggestion, I've heard that (and read it) as well for attempting to blunt subclone large fragments, and it is one more variation I will try...I am currently doing a 1:10 vector:insert using NEB Quik Ligase, that is done at room temp for a short period,and their buffer contains PEG,so we will see...
try increasing the DNA upto 250ng and use the NEB quick ligase kit. Also get some good cells. These might help you.
give it one last go with the blunt end startegy, follows scolix suggestion and increase the DNA concentration (ie use more DNA in the same volume). Dephosphorylate your vector. Also try PEG ligation at 4 degrees. Also see if you can do a kill digest a restriction site, to destory empty vectors, or vectors that have not been filled and simply religated to itself. And finally use some good company competent cell.
If that doesn't work. Change the strategy. fred_33's idea of introducing a MSC is a good idea.
As an option, another strategies you can do try is to PCR amplify your 6.1kb vector. All you will then need is to add the appropriate restriction sites to the amplification primers. Since the insert is excised by NheI and MluI, you will need restriction sites on the primers that make compatible ends (with the insert).
NheI compatible with SpeI / XbaI
MluI compatible with AscI, BssHII / AflIII (A/CGCGT)
Thus you can add either an NheI, SpeI or XbaI site at one end, and an MluI, AscI or BssHII, or AflIII site on the other. Pick the site which unique.
You will need a good proof reading polymerase to execute this proposal. (eg KODhifi)
i've tested myself pcr of vectors, and don't truly know what, but they never gave that good results. So i would do in parallel the vectors' pcr and an other strategy...
Hmmm.... have you tried linearising your vector/plasmid/template before amplification? I find that you get better yields that way.