GMT Tutorial Exercise 23 - Workflow 2 - Step 13.4
Prepare samples for DNA fragment analysis
Important:
- Once PCR products have been denatured in formamide (steps 2 and 3 below), 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.
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- The desalted PCR products have a 1/100 dilution. Dilute the PCR products further by adding an appropriate volume of sterile water as required for the DNA fragment analyser to be used, see the table below. Vortex the sample thoroughly and pulse spin. Typically, desalted PCR products with a final dilution of 1/125 to 1/500 are appropriate for analysis on a semi-automated DNA fragment analyser.
| DNA fragment analyser | Appropriate final dilution factor | Volume of sterile water to add to achieve final dilution factor
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| ABI 3730 | 1:250 | 1.5 x recovered volume of desalted PCR products
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| ABI 3700 | 1:250 | 1.5 x recovered volume of desalted PCR products
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| ABI 3100 | 1:250 | 1.5 x recovered volume of desalted PCR products
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| MegaBACE 1000 | (check with your service provider) | (check with your service provider)
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| MegaBACE 4000 | (check with your service provider) | (check with your service provider)
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Note:
The desalted PCR products can be recovered directly at the correct final dilution by adding an appropriately increased volume of sterile water when resuspending DNA from the AcroPrep 384 UF filter plate. The necessary volume of sterile water is calculated using the table for Step 4 of the instructions for desalting pooled PCR products and the final dilution factor from the table above.
- 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. For each well, mix:
| | 3 | | µL | pooled PCR products (desalted and at the required final dilution)
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| + | 8 | | µL | Hi-DiTM formamide
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| + | 0 | .4 | µL | GeneScanTM"“500 LIZ® size standard
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| = | 11 | .4 | µL | Total Volume
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Note:
GeneScanTM"“500 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.
- Heat the uncovered PCR plate at 90°C for 5 min to denature the DNA fragments and to evaporate the water component.
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