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Multiplex PCR - Multiplex PCR and gel electrophoresis (Sep/16/2009 )

Hi all

I am using Multiplex PCR (10plex assay) for typing and subtyping Influenza and Respiratory Syncytial Viruses. The method of detection used is Gel Electrophoresis. Can any one advice me on this?

1. What should be the ideal length range used for amplicon? Usually, I found 200-600 bp.

2. What should be the minimum and maximum size difference (in base pairs) between each amplicon considered for multiplexing?(In my case there will be 10 amplicons).

3. Is amplicon size difference varies with the type of electrophoresis?

4. What percentage of gel should I use for 10plex assay?

Mihika

-mihika-

mihika on Sep 16 2009, 11:06 PM said:

Hi all

I am using Multiplex PCR (10plex assay) for typing and subtyping Influenza and Respiratory Syncytial Viruses. The method of detection used is Gel Electrophoresis. Can any one advice me on this?

1. What should be the ideal length range used for amplicon? Usually, I found 200-600 bp.

2. What should be the minimum and maximum size difference (in base pairs) between each amplicon considered for multiplexing?(In my case there will be 10 amplicons).

3. Is amplicon size difference varies with the type of electrophoresis?

4. What percentage of gel should I use for 10plex assay?

Mihika


1. It depends on next question.
2. Minimum depends on gel resolution limit. For agarose ~25 for 100-300 b.p., ~50 for 300-500 and ~100 for 500-1000 (very rough estimate). Maximum depends on reaction speed and should not be > 1000 between smallest and largest product.
3. Yes, greatly. PAGE has a LOT more resolution ability (up to 1 nucleotide) than agarose gel.
4. Look answer before. It depends on what gel would you use.

-gleb.kudr-