38bp insert and 6.7kb vector ligation problem - the ligation efficiency is too low (Nov/19/2006 )
hi guys, I have some ligation problems now,
I am constructing a plasmid library, so I need a relatively high ligation efficiency, but the clones I get now is not enough, my quenstion is how to impove ligation efficiency. thanks
The process is as follows: we first synthesized 2 primers(by company)and the primers were random, after annealing and extention we got 50nt dsDNA, then we use sal1 and cla1 to cut them into 38nt dsDNA, we then tried qiagen nucleotide removal kit to extract this 38bp insert(although qiagen said the extraction range is 40bp-),after page gel identification, we confirm the extraction result, then we ligated this insert to the plasmid(6.7kb also cut by sal1 and cla1 ), the ratio is 10:1, we used takara ligase to perform ligation( plasmid 100ng) but the transformants are only 200 or so.
I need about 1000 clones /100ng, the low efficiency may be due to the random sequence or whatelse? could you please give me some suggestions? thanks a lot.
I don't know if I can improve this any, my bag of tricks for something this small isn't very good.
the only thing that comes to mind, would be to use more insert, and keeping the ligation mix as concentrated as possible ... ie more insert and vector in a smaller volume.
use of 10% PEG 6000 (magic ingrediant in quick ligase)... might help.
I was using molar ratios of 1:1 to 1:4 vector:insert with relatively good results, probably 500 to 1000 colonies when plating the whole heat shocked bateria.
I also ordered the primers alreasdy phosphorylated and complementary such that i dont have to digest them before ligation, that is.. living the respective overghangs free.
hope that helped a bit
You could prepare oligo directly for a 38 nt ds DNA, it don't require RE digestion and no 5' phosporated at both end, which would result in self ligation.
Self ligation might consume most of your dsDNA into a ligated products with only one RE site at both end (SalI :SalI or by ClaI:ClaI) if you did not dephoresphorated digested dsDNA and might resulted in low ligation efficiency to vector...
Thanks very much.
my problem is my primers are random, we have to anneal and extend them ,so we could just get blunt end dsDNA, it seems that we have to do enzyme cutting.
and after checking , I found PEG is for blunt end ligation only.
you might using Taq for extension, then result in products similar to PCR product and can be ligated into TA vector
the only thing that comes to mind, would be to use more insert, and keeping the ligation mix as concentrated as possible ... ie more insert and vector in a smaller volume.
use of 10% PEG 6000 (magic ingrediant in quick ligase)... might help.
Oh god, that what I want to ask for a long long time.
Promega ligase and fast ligation kit are just the same except the buffer of the fast ligation kit contain PEG.
At that time, I just don't know why should I pay for 100 US$ more for PEG containing buffer ?!
But, it really different without PEG.....how does it work ?
I don't know if I can improve this any, my bag of tricks for something this small isn't very good.
the only thing that comes to mind, would be to use more insert, and keeping the ligation mix as concentrated as possible ... ie more insert and vector in a smaller volume.
use of 10% PEG 6000 (magic ingrediant in quick ligase)... might help.
Oh god, that what I want to ask for a long long time.
Promega ligase and fast ligation kit are just the same except the buffer of the fast ligation kit contain PEG.
At that time, I just don't know why should I pay for 100 US$ more for PEG containing buffer ?!
But, it really different without PEG.....how does it work ?
No PEG6000.. and you don't have efficient ligation in under 30mins. Got to wait at least 2hrs...
5hrs and more for semi complete ligation.
The usual story goes something like this
PEG makes ligation go faster by taking up space. It cause DNA to be concentrated into a smaller volume thus increasing the effective concentration of DNA termini. In addition PEG somehow changes the physical structure of DNA... amount which is increase melting temperature. It is believed that this change in structure makes it more amicable to ligase binding....
This tale has not changed since its proposal in the early 1980s. I don't know if it is even right. It may be wrong but has since passed on to become the canon answer. I have not found any papers aside from these early, mid 1980 papers that study PEG and its effects on ligation.
hey, I am doing the same thing. My efficiency is also low. I posted a new topic there.