Hi. I am working on RAPD PCR of fungi. I have read papers on RAPD PCR and most of them mentioned that scoring is made on the basis of absence (0) or presence (1) of the band. Should the band size be determined as well? I understand that a ladder should be included when running the gel. So when you score the bands of the given sample, you should know the size. But what if the band is in between two known bands? For example, the band of your sample happens to be between 100 and 200 bp of a 100bp ladder. Can you just assume that it is, say 120 or 140 bp? Won't it be subjective and biased? I am confused over this 'cos someone told me that when I publish this, the reviewers might not approve my method.
Thank you very much in advance.
Hello,
in case of RAPD markers all amplification products (bands) that could be reliably read are treated as single dominant loci and scored either present (1) or absent (0) across genotypes. Based on that molecular data you will obtain a binary matrix which can be used for further studies. For the beginning I can recommend you GenAlEx software:
http://biology-assets.anu.edu.au/GenAlEx/Welcome.html
It is not necessary to estimate the size of each band in the band pattern obtained for particular sample. Usually this information is needed for some unique/rare alleles which determine special genetic features of a sample.
Application of DNA ladder in one of the lines on the gel will allow you to estimate the size of particular band. In order to estimate the molecular weight of selected band, first you have to plot a standard curve based on band pattern for the DNA ladder. This plot will visualize the relation between the size of the band and the migrated distance. Remember that DNA runs in a gel as a function of the logarithm of its molecular weight. When you already have the standard curve you need to measure the migration distance of "your" band and locate it on the standard curve - this will allow you to read the molecular weight directly from the plot (log scale).
There is also easier way to estimate the band size - there are some software packages which are able to do this automatically based on the reference of certain DNA ladder, but they are usually sold with the gel documentation station. Perhaps somebody in your Department/Institute have already such software?
Good luck
Piotr
Hello,
in case of RAPD markers all amplification products (bands) that could be reliably read are treated as single dominant loci and scored either present (1) or absent (0) across genotypes. Based on that molecular data you will obtain a binary matrix which can be used for further studies. For the beginning I can recommend you GenAlEx software:
http://biology-assets.anu.edu.au/GenAlEx/Welcome.html
It is not necessary to estimate the size of each band in the band pattern obtained for particular sample. Usually this information is needed for some unique/rare alleles which determine special genetic features of a sample.
Application of DNA ladder in one of the lines on the gel will allow you to estimate the size of particular band. In order to estimate the molecular weight of selected band, first you have to plot a standard curve based on band pattern for the DNA ladder. This plot will visualize the relation between the size of the band and the migrated distance. Remember that DNA runs in a gel as a function of the logarithm of its molecular weight. When you already have the standard curve you need to measure the migration distance of "your" band and locate it on the standard curve - this will allow you to read the molecular weight directly from the plot (log scale).
There is also easier way to estimate the band size - there are some software packages which are able to do this automatically based on the reference of certain DNA ladder, but they are usually sold with the gel documentation station. Perhaps somebody in your Department/Institute have already such software?
Good luck
Piotr
Thank you very much Piotr Androsiuk . I completely understand now how to analyse these bands. I sincerely appreciate it.
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