Which is the best way to visualize microsatellite bands which are ranging from 100 bp to 300 bp? while loading the PCR product on agarose gel with 1.5% concentration very faint and low quality bands are observed. What should be the percentage of agarose in the gel.Is there any other methods to visualize the bands? which type of Gene rulers/ladders that we have to use for clear visualization?
1.5 to 2% gels are fine and for good results should be run as slowly as possible to avoid heating which broadens the bands. Make sure the gel is well set and run at half the speed you usually do in 2%agarose. The problem is possibly in your pcr conditions.Can we see a picture of your gel please?
1.5 to 2% gels are fine and for good results should be run as slowly as possible to avoid heating which broadens the bands. Make sure the gel is well set and run at half the speed you usually do in 2%agarose. The problem is possibly in your pcr conditions.Can we see a picture of your gel please?
If you want to increase resolution of your polymorphic bands then you can add metaphor agarose or you should run gel on PAGE.
http://www.sciencedirect.com/science/article/pii/S2213596015001075
I agree with Kamal Kumar. Metaphor gels are very useful. Just be sure that PCR products are of high quality. If polymorphyc bands are very close each other, perhaps you could have probles to read the with accuracy, but you can increase the density of gels to 2.5% or 3%. Hope this helps.
High percent agarose, use the expensive kind that is designed for small fragments, and go low and slow on the voltage. Also, use just one comb per gel so you have a longer distance for your bands to travel. With long run times, adding ethidium bromide (or whatever dye you use) to the positive end of the gel tank will help keep the bands bright. Also, use a DNA ladder designed for small fragments.
Hi
PAGE with either silver staining or SYBR green DNA binding dye. Alternatively you may also use a gel-free analysis based on HRM (high resolution melting)
generally 1.5% -2% agarose gel are ok to visualize your concern band size. faint band often result from the improper PCR amplification. low quality band sometimes occur due to the variable ionic strength of the TAE buffer. if your ladder runs well then focus on the first problem. use of 100bp DNA ladder or 1Kb plus DNA ladder will be fine
Insufficient concentration of DNA as well as low volume of DNA added per well width may cause that. Your DNA might have been partially degraded. Kindly check your protocol and be sure you do not use nuclease specifically RNase. Another thing is you might have loaded part of your DNA off the gel mistakingly and subsequently electrophoresed off the gel. Sometimes, when you electrophorese the gel for less time, at a lower voltage, or in a higher percentage gel, it can cause the DNA to be missing on the gel. If you also stain your DNA with ETBR, you might have used improper UV light source. Pls check your protocol well and be sure the appropriate precautions are observed. Best wishes
Vijayan,
when we did microsat "by hand", we made migration in acrylamide gel. Highly resolutive, we read thousands of profiles with this ... But requiring specific material. So it depends that the purpose of your reading, just testing if the PCR works ? if so, likely no need to go with acrylamide, and agarose are said may be sufficient.
best,
Dear Keerthy
microsatellite bands must be analysis on 10% polyacrylamide gel (not agarose) and use Marker from 50 bp to 501 bp.
https://www.researchgate.net/profile/M_El-Sayed2/contributions
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