Mesoplasma florum:Electroporation

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
Line 23: Line 23:
* spin down again, remove supernatent
* spin down again, remove supernatent
* resuspend the pellet in the remaining liquid
* resuspend the pellet in the remaining liquid
-
* add 0.5 ml of chilled electroporation buffer, mix
+
* 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
* hold the cells on ice
-
* Aliquot 50 μl of cells into chilled eppendorfs
+
* Add 4 μl of transposome or plasmid DNA for transformation
-
* Add 1 μl of DNA for transformation
+
* Mix and transfer to chilled 1 mm gap electroporation cuvettes
-
* Mix and transfer to a chilled 1 mm gap electroporation cuvette
+
* Pulse the cuvette at 1.1  to 1.4 KV
-
* Pulse the cuvette at 1.2 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
+
* 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 2-3 hours
+
** cells can be held at 4° following outgrowth
** cells can be held at 4° following outgrowth
 +
* spin down cells, discard supernatent
 +
* resuspend in the remaining liquid, bring to 500 μl
* Plate 100 μl aliquots on selective plates
* Plate 100 μl aliquots on selective plates
-
* Transfer 100 μl aliquots into 10 ml of selective medium
 
* 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 13:55, 25 September 2007

Transformation of Mesoplasma florum by electroporation

Materials

  • overnight culture of Mesoplasma florum in ATCC 1161 medium
  • chilled mycoplasma electroporation buffer
    • 8 mM HEPES pH 7.4
    • 272 mM sucrose
  • chilled 1 mm electroporation cuvettes
  • chilled ATCC 1161 medium aliquots of 1 ml in 2 ml tubes
  • 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


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 1 mm gap electroporation cuvettes
  • Pulse the cuvette at 1.1 to 1.4 KV
  • Add 200 μl of chilled 1161 medium from a 10 ml tube, mix with the pipet
  • 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
    • cells can be held at 4° following outgrowth
  • spin down cells, discard supernatent
  • resuspend in the remaining liquid, bring to 500 μl
  • Plate 100 μl aliquots on selective plates
  • 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

Personal tools