I have prepared competent cells of L. lactis NZ9000. But transformation not working. No colony at all in the plate. Parameters are as following:
plasmid concentration: 1 uL (300ng)
Electroporator- Voltage 2.0, 1 pulse
Cells are viable (checked), Antibiotic conc. 10 ug/mL in plate
100 uL spread in one plate, Spin down...then rest of the cells spread in another plate.....no colonies.
Protocol followed:
1. refresh (test tube): 5 mL of GM17 liquid medium was inoculated with 1% L. lactis NZ9000 from glycerol stock and incubated at 30oC for 24 hours.
2. pre-culture (test tube): Two 5 mL bottles of GM17 liquid medium were inoculated with 1% of the refreshed culture and incubated at 30oC for 24 hours.
3. main culture (reagent bottle):
One thousand mL of SGM17 medium (42.5 g/L M17 broth, 102.7 g/L Sucrose, 10 g/L Glycine, 5 g/L Glucose) was inoculated with 1% of the pre-culture medium and incubated at 30oC until OD600 = 0.5 to 0.6.
4. the main culture was ice-cooled for 5 min.
5. The culture was transferred to four ice-cooled 500 mL centrifuge tubes and centrifuged at 4oC for 25 minutes at 5,480 x g. The bacteria were collected.
6. 40 mL of washing solution (10% (v/v) Glycerol, 136.9 g/L Sucrose) was added to each centrifuge tube and resuspended.
7. Each resuspension was transferred to ice-cooled 50 mL Falcon tubes, centrifuged at 4oC, 2,300 x g for 15 min, and the bacteria were collected.
8. 40 mL of washing solution was added to two of the four Falcon tubes and resuspended, then each was transferred to the other two tubes and resuspended again.
9. the bacteria were collected by centrifugation as in step 7.
10. 40 mL of washing solution was added to each of the two Falcon tubes and resuspended.
11. Centrifuged as in step 7 and collected the bacteria.
12. add 1.2 mL of wash solution to each of the two Falcon tubes and resuspend.
13. The resuspension solution was dispensed in 50 µL portions into PCR tubes arranged in a cooled PCR tube stand to make NZ9000 competent cells. After preparation, the cells were stored at -80oC.
What plasmid are you transforming with, how long are you allowing them to recover after electroporation without antibiotics before plating and what's the antibiotic?
Alexandra Johnson Thanks Alexandra.
plasmid is pMG36c. Recovery time ~4hours. Antibiotic is chloramphenicol.
You could try reducing the concentration of chloramphenicol down to 5 µg/mL, since L. lactis is pretty sensitive to it. If that doesn't work, I've had good luck with NZ9000 using the lithium acetate-DTT method:
https://doi.org/10.1186%2F1472-6750-7-15
The 10^10 CFU/mL concentration of L. lactis NZ9000 for the final cell density they mention is approximately an OD600 of 85 (calculated, from a 1:100 dilution). I can't remember if they mention it but you can definitely freeze aliquots instead of making the cells fresh every time. Also you can omit the antibiotics in the recovery broth, that part of the protocol never really made sense to me.
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