2 replies to this topic

[epicrazy]

For a certain pair of bisulphite primers, I get two different sized amplicons within different organs of the same animal. My expected amplicon ize is 330bp. I get this amplicon in most of my samples except a few including my methylated control. This different amplicon is about 60bp lesser. My samples are back from a c57 background whereas my methylated control which i bought is from balb c. Initially i thought the difference was because of the strain. But now im confused! As a control, i also the use the same primer set on genomic DNA to ensure there is no nonspecific binding. This is reproducible! I run my samples on a 2.5% gel and let it run for an hour and a half at 100V.
Im going to send them for sequencing, but any ideas meanwhile!!!??

[pcrman]

Quote

As a control, i also the use the same primer set on genomic DNA to ensure there is no nonspecific binding

How can you use the same primers to amplify genomic DNA (DNA w/o modification)? But it is a good idea to design a new set of genomic PCR primers (on unmodified DNA) to verify that BSP primers did not amplify the smaller amplicon by non-specific priming which is easier with modified DNA.

[epicrazy]

For the same gene I designed two BS primers. One set gave me the same sized amplicon with both bisulphite treated DNA and normal genomic DNA. When I sent this for sequencing, I got high similarity in sequence between the two and both being significantly similar to the converted sequence of my gene of interest! ( My water control was clean, this is bizzare to me also, there is no literature regarding a pseudogene).
So for the second set, I ran the genomic DNA control to make sure I wasnt picking up anything from unconverted DNA which will form part of the population in my bisulphite treated sample. Once this was clean, I proceeded to test all my different tissues (with now different sized amplicons.)
I will follow your suggestion and check my amplicons sizes again! THanks a lot!