Preparation of Cellular reagents from Plate Auto-induction media
1. Transfert a small lump of cell biomass into a fresh tube and freeze the rest at -20C.
2. Resuspend the cells into 1.2ml of Cold PBS and follow the original protocol for cellular reagents preparation from step6.2.9 (Protocol here). 3. Dilute resuspended cell pellet into cold PBS to obtain a suspension of A600 between 6.5 and 8
Measure A600 of a neat, 1:10 or 1:100 dilution. Multiply the value to get the actual final A600 number.
You might have to dilute several times before you get the right OD.
4. Calculate the volume of your final cell suspension to aliquot in each PCR tube
(that would contain 2 x 108 cells), using the equation:
Volume to Aliquot = 200/final A600 of cell suspension.
e.g. if your final A600 is 6.5, then
- volume containing 2x108 cells = 200/6.5
- volume containing 2x108 cells = 31µl
5. Aliquot either single reaction or 10X reactions worth of cellular reagents into 8-tube strips
of 0.2 ml PCR tubes e.g. using the example above, 3.1 µl (1x reaction) or 31 µl (10x);
6. Label tubes with reagent, date and operator
7. Incubate the tubes at 60C for 10min in a thermocycler or Heat block (heat treatment)
to make sure that produced cellular reagents are free from any living bacteria.
8. Place the tube strips with aliquoted cellular reagents carefully in a container 1/2 filled with desiccant,
leave tubes opened (using vacuum Tupperware is ideal).
9. Place the container overnight in a 37 °C static incubator.
10. After 18-24hrs check to see if the cellular reagents are completely dry.
Note: Leaving the reagents longer at 37 °C should not hurt their efficacy.
11. Once dry, close the lids and place them in a small bag at +4°C with a small amount of desiccant.