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.
- If the sequencing plate you need is not in BIO, you have to make your own.
- Remove two full tubes of Seq Mix from the freezer, along with Forward Primer (eg. LepR) and Reverse Primer (eg. LepF). If you have used mini primers, then use the full primers for sequencing.
- For the first plate, the forward reaction, you want to use forward primer (eg. LepF). Defrost the Seq Mix and the primer. The Seq Mix is 832 ul. Add 110 ul of primer to the Seq Mix tube.
- Take out a cold plate and put a non-skirted plate in it. Label it with the primer you have used.
- Pipette 117 ul into each of the first eight wells across the plate.
- Using a multi-channel pipette, add 9 ul to each well in the plate.
- If you are making a partial plate (ill advised, but sometimes there is no way around it), multiply the number of samples you have to sequence by the amount of Seq Mix and primer for each well. Each well contains:
- 1.1ul primer
- 8.32ul Seq Mix
- 8. For each PCR plate that you want to sequence, you have to make the two plates, forward and reverse.
- 9. Repeat the above steps with your Primer B.
- 10. Keep the plates frozen until you are ready to use them.
Next step is to follow the Sequencing Procedure For BIO.
Updated May 2 2009