Hi all,
I m very much confused for a double digestion reaction to check a fallout of 166 bp which has been cloned in 6900bp vector.
I have took around 1500 ng of my recombinant plasmid(6900+166 bp) as a template for a 20ul digestion reaction but after loading the complete reaction volume I didnt able to see any fallout in a 1% agarose gel . The vector backbone is quite bright and can be seen in a single band but thr is no band of 166 bp.
I have previously done colony PCR for my clone confirmation and it has shown a clear band of 166 bp.
any suggestion regarding this problem?
the amount of insert in your plasmid is 166 bases in about 7000 bases or about0.02 of the total dna is insert. As you added 1500ng of dna to the cutting reaction then 0.02 0f this is about 30 ng . Normally you would expect to see this amount with ETBR visualisation but it does depend how far you ran the gel ans the amount of diffusion of the band.Also on what dye you use for visualisation. Run the gel for only 10 minutes as 166 separates well from 7000 and you should see the band. Most dna over 20ng should be visible if not run too far. If this fails then digest twice as much and it should be easily seen. I am assuming that in your pcr the zero template control is blank so that you are not pcring a template that does not exist in your plasmid
the amount of insert in your plasmid is 166 bases in about 7000 bases or about0.02 of the total dna is insert. As you added 1500ng of dna to the cutting reaction then 0.02 0f this is about 30 ng . Normally you would expect to see this amount with ETBR visualisation but it does depend how far you ran the gel ans the amount of diffusion of the band.Also on what dye you use for visualisation. Run the gel for only 10 minutes as 166 separates well from 7000 and you should see the band. Most dna over 20ng should be visible if not run too far. If this fails then digest twice as much and it should be easily seen. I am assuming that in your pcr the zero template control is blank so that you are not pcring a template that does not exist in your plasmid
I agree with Paul. In addition, for my experience colony PCR is not always reliable. I suggest you confirm your cloning by performing PCR on the extracted plasmid or a better option is to digest your recombinant plasmid using an enzyme that targets the plasmid in the insert (an enzyme that dose not cut your plasmid). Then compare your linearized plasmid with control.
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