Mesoplasma florum:Electroporation

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* overnight culture of ''Mesoplasma florum'' in ATCC 1161 medium
* overnight culture of ''Mesoplasma florum'' in ATCC 1161 medium
 +
** preparing 10 ml cultures with 1, 10, 100, and 1000 μl of infected cultures assures that one of these will be ready the next day at the correct OD level.  Select cultures which are just changing color.
* chilled mycoplasma electroporation buffer
* chilled mycoplasma electroporation buffer
** 8 mM HEPES pH 7.4
** 8 mM HEPES pH 7.4
** 272 mM sucrose
** 272 mM sucrose
-
* chilled 1 mm electroporation cuvettes
+
* chilled 2 mm electroporation cuvettes
-
* chilled ATCC 1161 medium aliquots of 10 ml in 15 ml centrifuge tubes
+
* chilled ATCC 1161 medium aliquots of 1 ml in 2 ml eppendorfs
* Selective medium or plates
* Selective medium or plates
** Tetracycline resistance from TetM is higher than 200 μg/ml
** Tetracycline resistance from TetM is higher than 200 μg/ml
** Untransformed cells grow poorly at 4 μg/ml
** Untransformed cells grow poorly at 4 μg/ml
-
** Selective plates are at 10 μg/ml
+
** Selective plates are at 10 μg/ml or 20 μg/ml
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* hold the cells on ice
* hold the cells on ice
* Add 4 μl of transposome or plasmid DNA for transformation
* Add 4 μl of transposome or plasmid DNA for transformation
-
* Mix and transfer to chilled 1 mm gap electroporation cuvettes
+
* Mix and transfer to chilled 2 mm gap electroporation cuvettes
-
* Pulse the cuvette at 1.1  to 1.4 KV
+
* Pulse the cuvette at 2.5 KV
-
* Add 200 μl of chilled 1161 medium from a 10 ml tube, mix with the pipet
+
* Add 200 μl of chilled 1161 medium from a 2 ml tube, mix with the pipet
* Transfer the cuvette contents back into the same tube, repeat
* Transfer the cuvette contents back into the same tube, repeat
-
* Place the 10 ml tubes into the 30° incubator for outgrowth for 2-3 hours
+
* Place the 2 ml tubes into the 30° incubator for outgrowth for 3 hours
** cells can be held at 4° following outgrowth
** cells can be held at 4° following outgrowth
-
* spin down cells, discard supernatent
+
** dilute 1 μl of the culture into 1 ml of 1161 medium, vortex, and dilute again into 1 ml.  Plate 200 μl on a nonselective 1161 plate for counting untransformed CFUs.
-
* resuspend in the remaining liquid, bring to 500 μl
+
* Plate 200 μl aliquots on selective plates
-
* Plate 100 μl aliquots on selective plates
+
* Culture 200 μl in 10 ml selective liquid culture
* grow plates and medium at 30° for colonies or color change
* grow plates and medium at 30° for colonies or color change

Revision as of 20:41, 3 November 2007

Transformation of Mesoplasma florum by electroporation

Materials

  • overnight culture of Mesoplasma florum in ATCC 1161 medium
    • preparing 10 ml cultures with 1, 10, 100, and 1000 μl of infected cultures assures that one of these will be ready the next day at the correct OD level. Select cultures which are just changing color.
  • chilled mycoplasma electroporation buffer
    • 8 mM HEPES pH 7.4
    • 272 mM sucrose
  • chilled 2 mm electroporation cuvettes
  • chilled ATCC 1161 medium aliquots of 1 ml in 2 ml eppendorfs
  • Selective medium or plates
    • Tetracycline resistance from TetM is higher than 200 μg/ml
    • Untransformed cells grow poorly at 4 μg/ml
    • Selective plates are at 10 μg/ml or 20 μg/ml


Protocol

  • chill the centrifuge to 4°
  • spin down 10 ml of overnight culture at 8000g for 5 minutes, remove the supernatent
  • resuspend the pellet in the remaining liquid by vigorous vortexing
  • add 10 ml of chilled electroporation buffer and mix
  • spin down again, remove supernatent
  • resuspend the pellet in the remaining liquid
  • add 10 ml of chilled electroporation buffer
  • spin down again, remove supernatent
  • resuspend the pellet in the remaining liquid, bring the total volume to 200 μl
  • hold the cells on ice
  • Add 4 μl of transposome or plasmid DNA for transformation
  • Mix and transfer to chilled 2 mm gap electroporation cuvettes
  • Pulse the cuvette at 2.5 KV
  • Add 200 μl of chilled 1161 medium from a 2 ml tube, mix with the pipet
  • Transfer the cuvette contents back into the same tube, repeat
  • Place the 2 ml tubes into the 30° incubator for outgrowth for 3 hours
    • cells can be held at 4° following outgrowth
    • dilute 1 μl of the culture into 1 ml of 1161 medium, vortex, and dilute again into 1 ml. Plate 200 μl on a nonselective 1161 plate for counting untransformed CFUs.
  • Plate 200 μl aliquots on selective plates
  • Culture 200 μl in 10 ml selective liquid culture
  • grow plates and medium at 30° for colonies or color change


Notes 9/6/07: Arcing at 1.8 and 1.6 KV; possibly we need a second wash and/or some additional delay in the wash to remove more salt. Gel loading tips do not work. Plating 125 ul seems to work well. Tested outgrowth at 2 ug/ml, 4 ug/ml, 8 ug/ml

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