"
Team:Groningen/Protocols for Lactococcus
From 2010.igem.org
(Difference between revisions)
(New page: '''Preparation of cells''' *10 ml ON culture (SMGG) in 100 ml SMGG *Grow to OD600=0.2-0.7 *Wash three times with 50 ml icecold wash buffer*Resuspend in 1 ml wash buffer '''Electr...) |
|||
| Line 3: | Line 3: | ||
*10 ml ON culture ([[SMGG]]) in 100 ml SMGG | *10 ml ON culture ([[SMGG]]) in 100 ml SMGG | ||
*Grow to OD600=0.2-0.7 | *Grow to OD600=0.2-0.7 | ||
| - | *Wash three times with 50 ml icecold [[wash buffer]]*Resuspend in 1 ml wash buffer | + | *Wash three times with 50 ml icecold [[wash buffer]] |
| + | *Resuspend in 1 ml wash buffer | ||
'''Electroporation''' | '''Electroporation''' | ||
| Line 9: | Line 10: | ||
*1 μl DNA in 40 μl cell-suspension in ice cold cuvette | *1 μl DNA in 40 μl cell-suspension in ice cold cuvette | ||
*Electroporate at 2.5 kV, 25 μF, 200 Ohm. | *Electroporate at 2.5 kV, 25 μF, 200 Ohm. | ||
| - | *Add 4 ml [[SMG17MC]]*Incubate 2 hours (Ery induction=50 ng/ml) | + | *Add 4 ml [[SMG17MC]] |
| + | *Incubate 2 hours (Ery induction=50 ng/ml) | ||
*Concentrate cells to 0.5 ml. | *Concentrate cells to 0.5 ml. | ||
*Plate on [[GSM17- Agar]]. | *Plate on [[GSM17- Agar]]. | ||
Latest revision as of 20:54, 27 October 2010
Preparation of cells
- 10 ml ON culture (SMGG) in 100 ml SMGG
- Grow to OD600=0.2-0.7
- Wash three times with 50 ml icecold wash buffer
- Resuspend in 1 ml wash buffer
Electroporation
- 1 μl DNA in 40 μl cell-suspension in ice cold cuvette
- Electroporate at 2.5 kV, 25 μF, 200 Ohm.
- Add 4 ml SMG17MC
- Incubate 2 hours (Ery induction=50 ng/ml)
- Concentrate cells to 0.5 ml.
- Plate on GSM17- Agar.
