How can I separate PCR fragments that are small and very close in size on an agarose gel?

The concentration of the agarose gel for separation of multiplex PCR products should be appropriate for the overall size of products generated and can be adjusted for resolving small size differences between PCR fragments. For optimal results, we recommend the use of 1x TAE buffer for preparation and running of the gel. Use the general guidelines listed in the table below for choosing the percentage of agarose.

 

Minimum difference in size of PCR products	Maximum size of fragments	Concentration of agarose
>200 bp	2000 bp	1.3%
>100-200 bp	1000 bp	1.4-1.6%
>50-100 bp	750 bp	1.7-2.0%
20-50 bp	500 bp	2.5-3.0%
<20bp*	250 bp	3.0-4.0%

 

*Efficient separation of PCR products differing in size by about 20 bp is usually possible using standard molecular-biology–grade agarose. For separation of fragments that differ in size by less than 20 bp, we recommend using high-resolution agarose, for example MetaPhor® agarose (FMC Bioproducts). For more information, visit www.cambrex.com.

Please refer to the QIAGEN Multiplex PCR Handbook for additional information, and for details on successful multiplex PCR using the QIAGEN Multiplex PCR Kit.