Protocols for the Avant 3100 Genetic Analyzer

PCR for Fragment Analysis

Materials

The Vision lab typically mixes the forward and reverse primers to a final concentration of 10uM and adds 0.15-0.3ul/rxn to the PCR cocktail. If multiplexing, use the same amount of each individual primer and reduce the volume of water accordingly.

The thermal profile is 3 min at 94C, 25 cycles of 1 min 94C, 1 min at the primer-specific annealing temp, and 1 min at 72C, followed by 45 seconds at 55C and 30-60 min at 72C. The long final extension ensures that Taq adds a terminal adenine to *all* the PCR products. It is important to limit the number of cycles to 25, else the PCR product may need to be diluted 20-30X before being loaded on the sequencer.

PCR Cleanup for Sequencing

Do this prior to extension to remove excess dNTPs and unincorporated primers. Qiagen Kits work well if you have only a few samples. However, columns are expensive if you’re working with 96 well plates. Plus, the Exo-Sap method below is incredibly easy and works great for us!

Materials

Procedure


1. Amplify the desired PCR product.
2. Check the product on an agarose gel with a size standard. (This method will not ‘clean up’ reactions which yield more than one product).
3. If a single band has been obtained, then mix 5 ul PCR product with 2 ul Exo-Sap.
4. Incubate at 37oC for 15 min (chews up primers etc.)
5. Incubate at 80oC for 15 min (kills the enzymes)

Extension Reaction for Sequencing

For whole and half reactions, see the ABI recommended protocol. The following protocol is for 1/8th reactions, which we have found to be cheap and robust.

Materials

Procedure

Add ingredients to 96-well plate. Thermocycle program: 96C for 1 min, followed by 25 cycles of 96C 10 sec, 50C 5 sec and 60C 4 minutes. Hold at 4C.

IMPORTANT For some reason sequence quality may depend on setting a somewhat slow thermal ramp rate. This rate is adjustable on most new PCR machines. The Burch lab sets the thermal ramp to 1C/second.

Post-Extension Cleanup for Sequencing

ABI provides several options. The consensus from other labs is that the Centrisep columns (Princeton Separations) work best. This is an expensive option, especially when using 96 well plates. We are having good luck with ethanol precipitation. ABI provides two ethanol precipitation recipes. The Burch lab is currently using Recipe 2. The samples should be dried down in the 96-well plate in order to be submitted for sequencing.

Procedure

1. Make a master mix of one of the following recipes and add to each of the 10 ul extension products (double for 20ul):

Recipe 1 (supposed to give the cleanest results, but you may not be able to read as many as 50 bases following the primer; used by Vision lab)

Recipe 2 (better reads close to the primer; used by Burch lab)


2. Allow to sit at room temperature for 15 min.
3. Spin 96 well plates 4oC, at least 3,000 rpm, for 30 min.
4. Immediately invert the plate and spin 150 rpm, 30 sec to discard supernatant (no pellet is visible).
5. Add 50ul freshly-mixed 70% ethanol to each well.
6. Spin 96 well plates at 4 degrees celsius, 3,000 rpm, 15 min.
7. Immediately invert the plate and spin at ~750 rpm for about 30 seconds
8. Air-dry pellet for 60 min covered with foil, or vacuum dry

Foil covered plate is ready to be submitted.

Preparation of Fragment Analysis Samples for Loading

Per well

Mix as a cocktail and dispense 9.5ul per well.

Add 0.5ul (uncleaned) PCR product. Spin plate 30sec at ~3000rpm. Denature at 95oC for 5 min. Immediately place on ice. Store in freezer until picked up by operator. Plates can be stored frozen for at least 1 week without re-denaturation.