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Regriddng of clones for sequecing is simple but good records are required to ensure the sequenced clone can be traced back to the original DMSO archive clone. Clones can be identified by their microtitre plate address.
Suggestions to aid records:
Prepare a microtitre sized grid (Figure 1) and score the clones which have been selected for sequencing. The PCR microtitre plate can then be placed on top of this grid and the clones to be transferred easily identified. The clones have to be transfered to the fresh plate individually and it is easy to make mistakes.
FIGURE 1. Microtitre plate grid
1. Using a gilson pippette transfer 15ul PCR product of the selected clones 1 row (12 clones) at a time from the PCR plate to the regrid plate, leaving tips in the wells of the regrid plate as you work. This helps you identify which clones have been transferred. Again it helps to place a gilson pipete tip box insert ontop of the regrid plate to helps support the tips.
2. As you work mark the wells of the orignal PCR plate after every 12 clones have been tranferred. This allows you to identify the end of every row.
3. Keep a record of the original microtitre plate address of the first clone in the regrid plate and the last clone in the regrid plate. This helps identify the begining and end of each regrid plate.
Once the PCR procduct have been regridded they can be Shrimp Alkaline Phosphatase/Exonuclease I treated for sequencing or frozen until needed.
Shrimp Alkaline Phosphatase cleaning of PCR products for sequencing