NOTE: When you sequence, you sequence in both forward and reverse directions. But this is done with two different reactions. Each reaction should only have forward or reverse primer in it, not both. For one plate, two plates must be created for sequencing, one with the forward primer and one with the reverse primer.
- Use pre-made plates from BIO. You need to go to the DNA sequencing room, at the very back beside the thermocyclers there is a freezer. Make sure you get forward and reverse plates. Bring the sequencing plates back to the Science Complex. If you do not have pre-made sequencing plates, see Making Sequencing Plates for BIO Protocols.
- Centrifuge the PCR plate (this is the plate that you have already run the PCR reaction with and the E-gel image is on LIMS) for 20-30 sec.
- Centrifuge the sequencing plates for 15-20 sec. They should have barcodes on them and they should be on LIMS. See LIMS for Sequencing.
- Label self-adhering foil with the box number, the type of sequencing plate (eg. M13-F), your name, and the date. Put the sequencing plate in an empty plate (you could use the one that is used for balancing) and label the sequencing plate with the box number, the type of sequencing plate, and your name.
- Get a new box of 10ul filter tips. Add 1.5ul of undiluted PCR product for invertebrate or vertebrates using a 0.5-10ul multichannel pipette. Changing tips after every row.
- Seal the sequencing plate with the labeled foil. Cover the PCR product.
- Centrifuge the sequencing plate for 15-20 sec.
- Place it in the thermocycler and run the “SEQ31” program – for all kinds of sequencing reactions. Make sure to change “tubes” to “plate.”
- When the reaction is done (about 2 hours and 40 minutes later) put the plates into a black box because they are light sensitive and they will be destroyed otherwise.
- Bring them to BIO, into the DNA sequencing room and place them in the fridge, to your left as soon as you walk in the room, in either the box marked “Insects” or “Non-Insects” depending on what you have.
- You don’t have to fill out any forms for the Access System because you already checked off the box for sequencing back when you first submitted the form. If you did not fill out the form this way the first time, simply change the form on Dataserv and print a copy for the lab manager in BIO.
NOTE for Mini-Primers for Lepidoptera and sequencing:
- Use primer LepF on the PCR plate that has LepF/MLepR in it, and LepR on the PCR plate with LepR/MLepF in it.
- LepF is the forward plate, and LepR is the reverse plate.
Return to Barcoding in the Hanner Lab Wiki.
Updated May 15 2009