We need to grow 5ml of overnight culture according to the manufacturer. Then the protocol we have says to spin down the bacterial cells, but it doesn't mention the volume for reconstitution, is it 5ml of 20% glycerol/media?
This is the procedure I use for recombinant E. coli strains :
- Spin 2 x 2 ml of overnight culture in LB or 2YT in two 2-ml sterile Eppendorf tubes
- Discard the supernatant of the first tube
- Using a pipetting device, take out 1 ml of the supernatant and keep it inside the tip
- Discard the rest of the second supernatant
- Use the medium that you kept to resuspend and pool both pellets
- Transfer the suspension into a cryogenic tube containing 0,5 ml sterile 60 % glycerol => yields 20 % final.
- Mix well by pipetting up and down several times and store at -80 °C
When starting a new culture from the stock, don't thaw the stock, but scrape the frozen surface with a sterile toothpick and inoculate the medium with it.
It really depends on your downstream application. If you are putting away the cells for long term storage, you can add 5 ml and make 1ml aliquots to freeze. This bacteria concentration should be sufficient for streaking on agar plates in the future. If you are extracting something from the cells later, and hence need a higher concentration, then resuspend the whole pellet in 1ml.
20% meant for final concentration of glycerol in mixture. So if you want to make final volume 1 ml, you have to add glycerol 200 ul + 800 ul of re-suspended bottom part after centrifuge. If you want to make total 5 ml, glycerol 1 ml + 4 ml of re-suspended cells. Hope this helps.
- Badges
- Science method