I am trying to separate the SSR markers of Venturia Inaequalis (Apple Scab Fungus) having Bp range of 171 t0 180 (maximum difference of 1bp to 7 bp). But, while separating using Metaphor Agarose, it does not give clear difference of alleles.
Using 3% Metaphor gel you can clearly separate until 5bp differed alleles. Try to use a higher conc. (e.g., 4 or 5%), load higher amounts of PCR products (~20 mico or more) and run the electrophoresis for a longer time. If does not work, then you should load your probes on Polyacrylamide gel.
I agree thet using polyacrylamide gel can be necessary. Mix standard agarose with Metaphor agarose (1:1 or 2:1), your gel will be stronger and less crumbly.
Hi,
Earlier I did with metaphor agarose where I used to get 3 or 4 bands, but in Polyacrylamide gel , bands were differnciated very well. You may use PAGE gel , so number samples also you can add more..Let us know once you have used it.
The best advice here is to avoid doing this if possible - at the very least you should use polyacrylamide slab gels, and it would be best if you could use an old style sequencing gel rig and do something like silver staining. If you have to use a slab gel, then avoid lower order repeats (dinucleotides, trinucleotides) and focus on tetranucleotide repeats and higher - although these will tend to be less polymorphic. If you really only have access to agarose gel facilities I would consider using a marker type more suited to the technology, like CAPS.
Thanks Dan
I also tried PAGE 8% gel (denatured gel) in Sequencing unit (Genetix Brand) and after running for 2 hours at 45W, 1500V and silver staining I am confused which is my band of interest as I am getting may bands I tried to use ladder 10bp (Invitrogen) but that didn't work. Can you suggest me more.
Can I use native PAGE 8% and load the samples in sequencing unit and run it as indicated above or I should use denatured page only in sequencing unit
Make sure that you denature your DNA samples before loading into a denaturing PAGE gel. Also it helps to use a formaldehyde based loading dye. I use the biorad seqiGen GT rigs to run our ssr and run them at on the constant Volts setting at 1800V for about 3 hours sometimes more to get good separation of fragments. Of course if you are using a smaller rig (distance between electrodes) you should scale it down accordingly but if your voltage is too low secondary structure could cause aberrant band migration patterns.
You can run a non denaturing gel in a sequencing unit but in my experience the denaturing gels produce more consistent, and reproducible results.
Invotrogen sells an AFLP ladder that is best for running on these type of gels. But it's expensive.
http://www.lifetechnologies.com/order/catalog/product/10832012
Veronica's advice is spot on! I think denaturing gels are more consistent.
In relation to your multiple bands, there are probably two sources:
Firstly the downside with silver staining on a denaturing gel is that you generally visualise both denatured strands, so an SSR allele on a silver stained gel is frequently a doublet band.
Secondly, long perfect repeats often result in stutter bands, on either side of the allele. These will generally occur at regular intervals (equal to the repeat unit length) frequently getting more intense as you "approach" the real allele, and then decreasing in intensity as you pass the allele. Just treat the most intense band or doublet as the real allele. If you are working with a heterozygous diploid or polyploid the gels can become very confusing because of a mixture of multiple alleles, stutter bands and doublets, but once you recognise each of the features yo can eventually score the gels relatively easily. There are a bunch of things you can try to reduce the incidence of stutter bands (just google "reduce stutter bands microsatellites") but you'll probably always have some , so it's probably best to get used to dealing with them as an artefactual feature on your gels.
Hi. you can use Metaphore. Because it is fast and safe. For my opinion it is better than Acrylamide.
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