bren2010

As asked in the title ?

Can somebody please explain to me the difference between semi quantitative and quantitative PCR ?

Hi I wish to isolate eggs from sheep faeces and subsequently extract DNA and run a PCR from them. Part of the egg isolation technique involves floating them on a dense liquid. The three commonly used floatation liquids are Zinc sulfate, saturated sugar and saturated salt solutions. I was wondering what are the effects of these substances on the performance of the PCR reactions ?

After amplification of the intergenic spacer region (IGS) of the ribosomal DNA from nematode species by PCR, gel electrophoresis reveals multiple bands. Why is this ? Here is some information that may be relevant. Depending on the species four to five bands can be observed which can have different intensities. The top band is usually the most intense The primers used are designed based on the conserved regions of the flanking 18S and 28S. Stringent PCR conditions of 60 C annealing temp and low MgCl conc employed to reduce non specific amplification. The target is part of the rDNA which is a tandemly repeated unit. I have two lines of thought on why this is occurring. Initially I believed that the repeat copies of the IGS region were not homologous and therefore the primers produced multiple bands. However when I gel purified the most intense band and used this as a template in a PCR a complex banding pattern was also observed. So I now think that the primers are binding to areas within the target sequence. Any thoughts on why this is occurring would be very much appreciated.

Thanks very much to both of you guys, very informative and helpful.

I work with nematode (eukaryote) ribosomal DNA, In the literature I see that some times the 28S gene is refered to as the 26S gene or vice versa for different nematode species. Can anybody tell me whats the difference ?