Fragment Analysis of Microsatellites, AFLP, CRISPR and SHAPE Experiments
User performs PCR and prepares products for electrophoresis. It is the responsibility of the user to optimize the concentration of products in samples by first performing a dilution series (see below). We recommend performing PCR one marker at a time and then mixing fragments post-PCR “multi-plexing”. If samples are multiplexed post-PCR, users should mix fragments in the appropriate ratios before submitting. Keep in mind that for each additional marker added, all markers will be effectively diluted to 1/n of their original signal strength, where n is the number of markers added.
We recommend Qiagen Type-IT Master Mix for Fragment Analysis. There is magic fairy dust in the buffer and it can greatly improve results.
To find optimal intensities for each locus start with a dilution series for each marker or multiplexed set. Suggested protocol:
Column 1 (Wells A-D)—— 1ul of undiluted PCR, brought to total volume of 10ul
Column 1 (Wells E-H)—— 1ul of a 1:50 dilution of PCR, brought to total volume of 10ul
Column 2 (Wells A-D)—— 1ul of a 1:100 dilution of PCR, brought to total volume of 10ul
Column 2 (Wells E-H)—— 1ul of a 1:250 dilution of PCR, brought to total volume of 10ul
**For multiplexed combinations use 1ul of each PCR product and bring to total volume of 10ul
Preparing Samples with your size standard
Preparing Samples for us to add size standard
|1 µl….Product (more or less, determine by dilution series)||1 µl….Product (more or less, determine by dilution series)|
|X µl….Hi-Di Formamide or molecular grade water||x µl….Hi-Di Formamide or molecular grade water|
0.5 µl-1.5ul….Size Standard (depends on the standard
contact us if you have questions.)
|15.0 µl Total Volume||10 µl Total Volume|