Hi.I am facing problems in the bacmid isolation step.Currently i am using purelink hipure plasmid miniprep kit for isolating the bacmid.But the problem is,after the isopropanol-ethanol step when i try to dissolve the bacmid pellet in TE buffer,it is not getting dissolved.First i thought i have over dried the pellet.So i tried to do the isolation again.this time i was very careful and i am sure the pellet was not over dried.but again it is not getting dissolved in TE.I tried using the kit TE and also the freshly prepared TE.I even tried keeping the pellet at 4 degree,37 degree and all that.But the pellet is not just getting dissolved.
is it the problem with the kit? any suggestions?
Dear Harsha
- the problem is likely to be contaminants; usually protein bleeding through into your final pellet
- I havn't used your particular kit but have performed many plasmid preps with a variety of extraction kits over the years
- In the first instance, I would try lysing your prep in double the amount of buffer and if not doing so leaving lysate on ice for 10 min on ice
- Furthermore, having lysed your cell Im assuming there is a step in your kit (usually y 2-3) where you add a solution and you obtain white flocculate precipitate ? This is when you remove most of your protein entangled with host genomic DNA leaving just a clear lysate containing your plasmid plus salt
- To more effectively remove protein which might be leading to your insoluble pellet at the end
- Proceed as described in manual with standard purification method
- There are other tricks you can deploy but start with that one and I can if necessary advise you further
Dear Harsha
- the problem is likely to be contaminants; usually protein bleeding through into your final pellet
- I havn't used your particular kit but have performed many plasmid preps with a variety of extraction kits over the years
- In the first instance, I would try lysing your prep in double the amount of buffer and if not doing so leaving lysate on ice for 10 min on ice
- Furthermore, having lysed your cell Im assuming there is a step in your kit (usually y 2-3) where you add a solution and you obtain white flocculate precipitate ? This is when you remove most of your protein entangled with host genomic DNA leaving just a clear lysate containing your plasmid plus salt
- To more effectively remove protein which might be leading to your insoluble pellet at the end
- Proceed as described in manual with standard purification method
- There are other tricks you can deploy but start with that one and I can if necessary advise you further
Solubility of "extracted" plasmid DNA depends upon a number of factors, including the GC content of the plasmid DNA. To start with, gradually increase NaCl concentration in the TE to dissolve the DNA. It may take more than 24 h at 37 degree C to dissolve most of the DNA. If this modification does not work, it is likely that you have non-soluble pellet that may contain non-nucleic acid material(s). In this case, what Laurence has noted above should work.
Dear Harsha,
I agree with the answers and suggestions given above. However, I need to raise some questions for you. Did you run the supernatant after dissolution of the pellet in TE buffer on agarose gel for detection of your target DNA? Did you try to dissolve the pellet using nuclease-free water instead of TE buffer?
Thank you for the suggestions.I will try the above said suggestions and let you know the results.
@ Alu Tekewe, yes I tried using nuclease free water but haven't tried running the supernatant on agarose gel yet.will do that today.
Thank you
@Laurence Stuart Dawkins-Hall..I tried all the things you have suggested..but unfortunately it s again the same.peller it's not getting dissolved.i have isolated plasmids before and have never faced such issues.but not getting the same results with bacmid.
@Divaker choubey I tried to increase the concentration of TE but still no hope
Dear Harsha
in these situations, sometimes double column purification can slightly diminish yield but increase purity resulting in pellets that dissolve. To reiterate, pure DNA will always dissolve in TE unless over concentrated in which case it dissolves but is highly viscuous
As others have pointed out, it is likely that the DNA is contaminated with free protein and/or DNA-protein complexes of genomic DNA. In this case it is worth doing a midi prep procedure to increase volumes and yield. Then, after you spin out the white precipitate from the neutralisation step, filter the supernatant through a whatman 1 paper into the column and wash as normal. This takes out residual protein complexes. When DNA is pure, pellets from precipitation/centrifugation should be invisible and should dissolve readily in aqueous buffers.
Good luck!
Harsha, I think there is no problem, I faced such like your results but for another aspect.
- I'm not sure about the kit component, but it maybe content glycogen solutions. Firstly try to reduce the volume isopropanol-ethanol, (particularly ethanol amount per sample)/ (plasmid concentration is low vs the reagent volume per sample).
- Try to reduce centrifuge time and it is better to be gravity force X g.
- If you get similar results don't care, then measure the concentration and the quality of the pure solution after shaken and vortex.
Anyway, Qiagen Large Construct Kit worked wonders.
@Ammar Elakhdar Thank you for the suggestions !
I am planning to buy that kit anyways ! Some else also suggested me that kit.!
I will try that and let you know the results :)
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