Hi all I have a full length (FL) sequence gene and few constructs of the gene to produce a functional protein.. which I would purify if all goes well.
Firstly, I did normal cloning of my FL gene and its constructs i.e. Molecular cloning of them using Pcold vector TF and Prs2.. It seemed positive till now and I have send the clones that were positive for sanger sequencing (did mini prep etc for the same using Kit) awaiting results.
Now the second part which is that I tried LIC and ran gels I obtained faint bands which was of the primers and not the desired sequence.
Tried with optimizing the PCR by adding DMSO.
Please suggest how could solve this problem.
Regards,
Ratish Raman
I think I didn't get the point.
You have plasmids containing your FL gene and some other constructs already and sent them for sequencing, so everything's fine?!
Why do you want to do LIC as well? When you have all your clones, then it's fine.
However, try to digest your clones with a restriction enzyme of choice to verify the plasmid's identity (use for example ApE to digest your plasmid in silico to see what bands should appear). You should choose a restriction enzyme with 4 to 8 digestion sites. Normally we do that before sequencing, because it's faster and cheaper. In general, an incorrect / shuffled plasmid can disturb the priming sites for your primers and thus, you won't get any product in your PCR.
Have you optimized the PCR conditions for your LIC. Just check the annealing temperatures of the primers and perform the PCR using those conditions. If it does not work use PCR enhancers like Solution-Q (Qiagen) PCR enhancer from Invitrogen. It might work.
sagar... the PCR was optimised with DMSO. but it didnot work. could u suggest any other I was thinking about MgSO4 or including betaine? is it a good choice?
Andreas..hey see I did a normal cloning as we do and then proceeded with t4 transformation. well all these went well. now then we wanted to do LIC and check but it didnot work.. so I wanted some tips and guidance on how to optimise the conditions for LIC.
I generally work with PCR enchancers from Qiagen or Invitrogen, as it is less of a hassle. Check if the primers annealing temperature experimentally, because it can be lower than what you have calculated. Also check for if there are serine, leucine or arginine residues in the sequence you are amplifying, if you do, then you will have to redesign your primers.
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