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...)
 
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*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'''
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*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.