Problem with cloningNde1-Not1 digest
Posted 27 May 2013 - 08:57 AM
I have been using pET28a vector for cloning. I have digested it with Nde1- Not1 overnight, performed the ligation and transformed in DH5alpha. However I have either got only pET28a colonies or no colonies at all. When the ligation reaction reaction was loaded on agarose gel, the insert remained hence no ligation was achieved. I have used a fresh ligase so no question of it not working. Does anyone have any experience with this combination of restriction enzymes? If so could u provide the optimum incubation time for this combination. Thanks in advance.
- TechnoJr likes this
Posted 27 May 2013 - 11:29 AM
I always had an alternate hypothesis....
Posted 27 May 2013 - 11:42 AM
Well I have compared the uncut and uncut plasmid and I cut see the linearized vector. I am however unsure if overnight digestion has to done for Nde1-Not 1 combination. I have included a positive control i.e. only pET28a and got a good transformation efficieny so no problems with that. I have to work on two halves 1. Standardizing incubation time 2. Ligation. So I would like to know how long should one go for Nde1- Not 1 digestion
Posted 27 May 2013 - 12:23 PM
Posted 28 May 2013 - 09:15 AM
What I wanted to ask was just that how much hour incubation would be sufficient for Nde1-Not 1 digestion.I went for an o/n incubation and read somewhere that 1-4 hour incubation was enough. So any experience so far on this combination i.e. Nde1 - Not1 digestion?
Posted 09 June 2013 - 11:39 AM
Posted 16 June 2013 - 09:00 PM
I don't have experience with those specific restriction enzymes but I have experience with double digestions. I prefer the lower concentration of enzyme with a longer incubation time (i.e. O/N).
Also just to be on the safe side, you can try individual digestions. Write down your digestion conditions for each enzyme, then perform them separately. Then you can confirm that your digestion
conditions indeed work by visualizing that each enzyme linearizes the plasmid vs uncut plasmid. Then you can be confident that both enzymes will cut following those digestion conditions.
Posted 17 June 2013 - 08:22 AM