Problem with cloning - (May/27/2013 )

Hi everyone,

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.
Manavi

you might hav to give more info about how much vector/insert and enzyme used for digestion. what evidence you have that both the enzymes did complete digestion? vector, insert ratio used?size of your insert? did you run vector ligation along with the vector-insert ligation on agarose? what abt the efficiency of your competent cells?

Hi GNANA,
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

Your problem is unlikely to be ligation. Where does the insert DNA come from?

Hi
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?

Thanks
Manavi

I don't have experience with those enzymes specifically, but my default is an overnight ligation at 16C. Also, make sure you digested your insert with those enzymes to make sure those ends are sticky too.

Hi Manavi,

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.

Best,
Ted

Hi thiong..well I got my clone.. n for the benefit of everyone ..I just did 4 hours of Nde1 and Not1 double digest in buffer 3. The problem was the bad quality of the vector before. So thanks everyone for your suggestions