I am trying to ligate an insert into a vector using 5' EcoN1 and 3' EcoR1 sites. These are realistically the only sites I can use to get the insert in.
After cutting with EcoN1 I am left with a single A/T overhang on my insert + vector. I tried ligating at room temp for 30 min and got zero bacterial colonies growing on selective media (whereas I get loads with other enzyme ligations). I am thinking of trying 16oC ON to see if this improves matters. I always add a massive excess of insert compared to vector DNA and it seems to work most of the time. I also tried a 3:1 ratio (volumes) of insert:vector alongside and this gave zero colonies also.
Anybody with suggestions/experience with EcoN1 ligations?
You might want to check this URL
http://www.neb.com/nebecomm/products/productr0521.asp#
It says...
General notes:
EcoNI produces DNA fragments that have a single-base 5´ extension which are more difficult to ligate than blunt-ended fragments.
Yes I had read that - doesnt have much in the way of suggestions on what to try so thought I would throw it out there as there are examples in the literature that have used EcoN1 + another enzyme to clone into vectors
This is an old observation, the standard way is to blunt the fragments. This can be done with the Klenow fragment in two ways, since you have a 5' overhang. You can either remove the overhang using Klenow in the absence of dNTP using the 5'->3' exonuclease activity or fill the overhang by adding dNTP's and continue with blunt-end ligation procedure.
Ole Skovgaard's answer sounds optimal for increasing the probability of ligation. Enzyme like sac I have 3' over hang could be compatible with Eco NI that has 5' over hang provided the bases for 5' and 3' over hang are complementary. Easier procedure would be to blunt end.
Have you checked that you DNA ligase can work/or the competency of your cells? Adding some PEG often helps in blunt end cloning maybe you can try it. Overnight ligation at 4 degree works pretty much for me as well.
The best way is to blunt it. But also you can try to dephosphorylate 5' ends of vector with BAP or CIAP, maybe it will help...
I am just wondering how you get the A/T overhung insert (not plasmid). If the overhung bases do not make a pair (A:T pair in this case) at the connection where ligase works, it would be extremely difficult to ligate; I would say impossible. You might want to make blunt ends prior to ligation.
Others have already suggested it, but I wanted to share that I personally have had success ligating an EcoNI-cut end to a BamHI-cut end after blunting with Klenow. Ligated for only 15 min at RT, nothing special on that end. Worked great!
I got some colonies in my insert + vector ligation when I left the ligation of the cut vector/insert overnight at 16oC (none in vector only control). Im currently screening to see if they are right.
If you are sure that your inert and vector EcoNI cleavage generate compatible overhangs (which is not always the case given the recognition sequence CCTnn/nnnAGG), you can try using the ligation buffer with PEG at room temperature. Fermentas sells the 10x ligase buffer and 40% PEG, you just have to mix these ingredient in 1:1 ratio to obtain an excellent 5x ligation buffer.
I was using it with overnight at 16oC and it was al right.
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