GMT Tutorial Exercise 21 - Workflow 1 - Step 8.3
Prepare samples for DNA fragment analysis
Important:
- This final step ensures that the PCR products are loaded onto the DNA fragment analyser under denaturing conditions.
- Once PCR products have been denatured in formamide, they should be analysed as soon as possible on a DNA fragment analyser. For best results, this should occur within 4 hrs and no longer than 12 hours after samples have been prepared.
- Given a DNA fragment separator's run times, this would mean e.g. checking before denaturing samples that a 384-well plate can be loaded onto the machine immediately afterwards. This can also restrict the number of plates that can be submitted at a time.
- Contact your DNA fragment separation service provider for details and to book a run.
|
- In the wells of a semi-skirted 96-well PCR plate, combine the desalted and correctly diluted PCR products with fluorescent dye and denaturant for capillary separation. This and the previous pooling and dilution steps will result in a 100-fold dilution for each marker. For each well, mix:
| | 3 | | µL | pooled PCR products
|
| + | 8 | | µL | Hi-DiTM formamide / GeneScanTM500 LIZ® size standard Mix (see below)
|
| = | 11 | | µL | Total Volume
|
Hi-DiTM formamide / GeneScanTM500 LIZ® size standard Mix
- For a 96-well plate:
Add 4 µL GeneScanTM500 LIZ® size standard to 800 µL Hi-DiTM formamide. Vortex and pulse-spin before aliquoting 8 µL to each well.
- For a 384-well plate:
Add 16 µL GeneScanTM500 LIZ® size standard to 3200 µL Hi-DiTM formamide. Vortex and pulse-spin before aliquoting 8 µL to each well.
- Heat the (covered) PCR plate at 90°C for 5 min to denature the DNA fragments and to evaporate the water component.
Note:
GeneScanTM500 LIZ® size standard and Hi-DiTM formamide can be purchased from Applied Biosystems. It is important that Hi-DiTM formamide is stored in small aliquots at -20°C in a constant-temperature freezer to prevent ionization, which results in significant loss of genotyping quality.
|
