that will not run through agarose gel about 75% of the time. We've gone back to check the primers and adjusted
I have had this happen in three out of four different samples; only with this particular sequence. It is supposed to run out to a similar length, about 250bp, to the others in this particular gel.
try running it a much longer time with a 1Kb marker, see how big it really is. it might be a chain made of different cDNA pieces. can you start over?
I have started over four times, but I'm using the same primers and template in each. I rechecked my oligos and our PI altered annealing/melting temps. I also tweaked my DNA/primer ratio. I may run it out after only 10 cycles or so in the PCR and see how big the product is.
Matt, try loading least amount of your PCR product, or try reducing the gel percentage...what i think it is a problem of some protein precipitation with your DNA...or may be over amplification of your template DNA to yield a longer than 250 bp desired sequence. it would be better if your dilute that particular PCR product before loading.. doing so may give you an insight on what may be happenning.
Looks like a huge piece of DNA? genomic? Run much longer. Your primers are not giving the expected size. OR cDNA did not amplify. Run RNA on gel before you do the cDNA reaction.
The marker run looks good. I would try to diluite the PCR product as others said.
I understand that this particular samples gave a band of normal size in some experiments... Am I right? But, still, they are prone to give this super-sized product...
No, in some I don't get any result at all. At this point I'm starting over with two new sets of primers in different locations on the gene. I just need a probe that is 8-10% of the total size of the gene.
Sure, maybe this option will save you time.
Anyway, the result is VERY strange because of the enormous size of the product that should be very difficult to obtain (it is a single defined band, not a smear) by PCR…
Good luck
Yes, it is odd. I've not be able to determine how large it is by running it out. Concentrations are the same as other probes that worked but it is too massive to get any discernible movement in gel.
Looks like you don't have amplification of your target in that lane. If you started with total nucleic acid then DNAse treat your extracts before cDNA synthesis
I guess, the problem is with intial DNAse and protein tratment , do both to your starting total RNA , then amplify it..take a serial dilution of your RNA and see if the band intensity diffres, if its a cDNA amplification , it would surely differ with your dilution but, as I suppose, its untreated RNA , or arather the genomic DNA contamination..check your primers by homology search if, it amplifies a very non specific areas of genome it can give a larger fragment !! but I still opt for my first answer..good luck!!!
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