Sequencing

The old-fashioned way of Sanger sequencing requires three steps: First you PCR-amplify your fragment of choice, then you run a sequencing reaction, and finally you visualize your work in an automated sequencer.

PCR

A reaction volume of 20 μl is sufficient. Make sure that your reaction yields a single band, otherwise you will have to resort to gel extraction to separate the band you are interested in.

Cleaning up the PCR with Exo-SAP

1. You need: Exonuclease I, SAP, and ddH₂O. Note: Promega does not sell SAP anymore. They now have TSAP and I guess it works equally well.
2. Prepare a master mix of ExoSAP to be used with the PCR products. It is a good idea to make a little bit too much (+ 10%), just in case. Prepare the master mix immediately before use and keep it on ice. Remove enzymes from the freezer immediately before use and keep them in an insulated container. Return the enzymes to the freezer immediately after use.

1. Add 5 µL of master mix to 11 µL of the PCR sample.
2. Incubate samples at 37˚C for 30 minutes and then 95˚C for 5 minutes in a PCR machine.
3. Store samples at 4 ˚C or -20 ˚C until needed.

Hint: SAP tends to become inactivated after prolonged storage (a few months) at -20 °C. If you don't use it regularly, aliquot it and store the aliquots at -70 °C.

Sequencing reaction

Reaction clean-up with a sephadex plate

1. Add SephadexG-50 to each well of Filterplate MSHVN4510 (Millipore). We have some tools ready in lab J18 that can be used to do this.
2. Place the filterplate on top of a collection plate, which is simply a 96-well plate.
3. Add 300 µL of distilled water to those wells you intend to use (one filterplate can be used for several runs) and let it soak 2-3 hrs at room temperature (or at 4°C over night).
4. Centrifuge the plate for 4 min at 2300 rpm and empty the collection plate.
5. Add 150 µL distilled water and centrifuge again for 4 min at 2300 rpm.
6. Now replace collection plate with the plate that goes into the sequencer (AB1100) and load the reaction on the sephadex in the wells.
7. Centrifuge for 4 min at 3500 rpm.
8. Place a septa mat on top of the plate and you are ready for sequencing.

Sequencing on ABI 3130XL

The 3130XL can run 16 samples at a time. They must be in two adjacent columns in your plate. If that's the case, and if you have reserved a time slot for the sequencing, go down to the GDC.

First connect to the NAS on which you have saved the information about your run. This is done via "map network drive". Note that you must write your username as follows: "d\username".

Second, open the software "Foundation Data Collection", which is used to run the sequencer. Go to the "plate manager" and import your .txt file that you prepared for the run.

Now go to the "run scheduler", set it to "advanced" and search for your file. Once you have located it, it's time to load the plate into the machine. Pack it into the carrier tray, place the cover on top and place the whole thing in the tray of the machine. If you do it the right way round, you will see that the image of the tray in the "run scheduler" turns to yellow. Now you can link your file to that tray. Check the "run view" if everything is alright (if you have only 16 samples, your file should also contain only 16 rows - you may have to edit that if you didn't before).

Now you can run the sequencer. The output files will be on drive E in the corresponding folder.