Ligationdifficulty - (Jul/26/2007 )
Hi there.
I am trying to transform DH5alpha with a plasmid containing an insert between NdeI and BamHI sites. This is the first time i am using NdeI. I am not able to get any positive colonies. I have been told that NdeI is not fun to work with. I am wondering if someone can please give me advice on getting this ligation!! I am digesting both fragment and plasmid for 1 hour at 37°C with excess NdeI then de-phosphorylating the plasmid. I have attempted ligation at both 4°C overnight and room temp for 4 hours. both with no luck.
I would really really appreciate anyones advice and tips on getting this to work.
Thanks heaps in advance!!!
Hmm... NdeI is not the fastest enzyme. NdeI needs at least 8bp around its site to cut. How far apart are the BamHI and NdeI site? Can you confirm that the insert and vector have been digested? How about cutting your DNA first with NdeI, followed by BamHI. Alternative, can you leave your digest cutting longer? How much DNA are you cutting and how much enzyme are you using? Depending on the numbers, you may not be giving your DNA enough time to digest.
Where does your insert come from? if PCR, there is the danger of the NdeI site
Over dephosphorylation is a problem. What are your conditions?
Where does your insert come from? if PCR, there is the danger of the NdeI site
Over dephosphorylation is a problem. What are your conditions?
Wow!! thanks for your reply! many questions!! hehe well, the 2 sites are far enuf apart in both the plasmid and the fragment. What i think i will attempt now is digesting with NdeI first, check it on gel, then digest with BamHI if the NdeI linearlises the plasmid. Thank you so much for your reply!
The issue may also be with your insert and not your plasmid. So you'll need some extended sequence on each end of your insert as well to accomodate the RE digest.
I would also think that the problem is digesting the insert rather than the plasmid. With the insert are you trying directly digest a linear PCR product? This could be tricky with NdeI but doable.
What worked for me is topping up the NdeI concentration during a digestion over a few hours.
If your insert is a PCR product you could subclone it into another vector such as pET151-D. This is straight forward and no digestion is necessary. You would probably have to redesign your forward primer for obtaining your insert though (and the reverse if you were to subclone into a different vector with the conventional multiple cloning site).
Someone suggested over dephosphorylating your plasmid could be a problem. I cannot see why this would be an issue if you are only dephosphorylating the plasmid and not the insert.
WIth tricky ligations I tend perform a PCR clean-up on the samples to be ligated and in the final step I elute with warm water.
The ratio of insert to plasmid can also be critical with tricky ligations especially where NdeI is concerned as from my experience you get very little digested is very inefficient
Sat
cheers sat. sounds like you know what your talking about with that NdeI. i will try as you suggest to re-digest the fragment for longer and more NdeI.
thanks again