Published in bio.philica.com
Microsatellite set up and analysis for multiplex specimens can be a challenging task. The following discussion is based on experiments I have performed on the ABI PRISM 3100 Genetic Analyzer.
1) Have 1 set of PCR samples where either the size is known or if not known, there will be the basis of this experiment.
2) The unknown were in 2 seperate PCRs with different cycling conditions ( if the size is x<200, cycle higher, I suggest 60-65 cycles. If the size is larger x>200, then cycle from 30-45; between 45-60 is where the tweaking takes place). Below is the cycling conditions I used for my multiplexing experiments. The size of my fragments were anywhere from 110 - 300.
Initial denaturation 94°C 2 min
Denaturation 94°C 15 s – 30 s
Annealing 50° – 65°C 30 s – 60 s
Elongation 72°C 45 s – 3 min
(above for for 45 – 60 cycles)
Final elongation 72°C 7 min
This way, one can tell from one 96 well plate what conditions work best. 9-13 primers are a lot, It will be hard to make sense of it all especially if it is all in one well. I know in my experiment, I was also using the multiplexing as a form of quanitative PCR, i.e if a well where more than one DNA type and primer existed, if a signal does not come up for the given sample where it is expected it could either be:
1) masking the sample which would mean many things depending on what one is looking for:
2) a frame shift occur which means a mutation is possible and needs to be compared to standard frames +2,+2, +3, -1,-2,-3 for mitchondrial of the given organism being tested.
The above situations can be confusing if this user does not have the microsatellite analysis results for the control samples.
The above situations can be confusing if the technician does not have the microsatellite analysis results for the control samples.
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This Observation was published on 10th August, 2006 at 00:58:46 and has been viewed 8738 times.
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The full citation for this Observation is:|
Mitchell, M. (2006). Microsatellite set up and analysis for multiplex specimens using an ABI PRISM 3100 Genetic Analyzer. PHILICA.COM Observation number 21.