Dear colleagues,
I am in trouble to extract sufficient DNA from the Clonewell E gel.
I have an 11 KB plasmid and want to purify it after restriction enzyme digestion to get rid of a 2.3 KB fragment.
Once I finished the double digestion, the sample should become two fragments: 9 KB desirable linearised vector (the one I want to extract) and 2.3 KB undesirable fragment.
I loaded about 240 ng DNA at my 1st attempt. I could see a clear band on the gel, but it just become wired once it entred into the collection well, I could not see it clearly in the collection well. And once I extracted it the yield was very low, only 0.3 ng/ul.
I know E gel Clonewell recommends to extract DNA upto 6 KB, but as I do not have traditional gel at the lab now, and it took time to get new ordering, I wonder it Clonewell might work in my case.
Anyone has similar experience to extract large DNA fragment from Clonewell Gel? What is the recovery rate you had?
Many thanks for your help.
Kind regards,
Rex
I have tried using clonewell e gel 0.8% gels for seperating some DNA bands a 700bp fragment from a 1000 bp fragment and and I seem to have ran into your same issue the gel starts off fine for the first few minutes but then it goes rubbish after this and everything becomes a mess on the gel and its very difficult to see when the band is in the zone. I will be going back to the old school method of making my own gels and cutting the bands out. I would be interested to hear if any has been able to get success with the clonewell gels maybe there are some tips and tricks that I am missing
Peter Durcan Hi Peter, thanks for your answer, I actually did extract some DNA from 0.8 % E clone gel eventually!!! What I did is just run the gel back and forth so that I can get as much DNA as possible. I ended up getting about 1mL solution with a very very low concentration (about 0.7 ng/ul). I had to use vacuum dryer to make it more concentrated, but I did successfully use it to conduct the Gibson, and the gene sequencing test went back perfect.
But yeah, I went back to the conventional method after that nightmare. It took too much time and effort. The Thermo Fisher ad tells you that it saves the time from gel purification, but you will spend more time wait the band reach into the column, so it does not save any time at all.
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