Mesoplasma florum:Electroporation

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
Current revision (19:22, 20 December 2010) (view source)
(Materials)
 
(9 intermediate revisions not shown.)
Line 1: Line 1:
 +
{{back to protocols}}
Transformation of ''Mesoplasma florum'' by electroporation
Transformation of ''Mesoplasma florum'' by electroporation
-
Materials
+
==Materials==
* 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 (93.1 g/l)
* chilled 1 mm electroporation cuvettes
* chilled 1 mm electroporation cuvettes
-
* chilled ATCC 1161 medium aliquots of 1 ml in 2 ml tubes
+
* 10 ml chilled ATCC 1161 medium
* Selective medium or plates
* Selective medium or plates
-
** Tetracycline resistance from TetM is higher than 100 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
+
** Tet selective plates are at 15 μg/ml
-
 
+
==Protocol==
-
Protocol
+
* chill the centrifuge to 4°
* chill the centrifuge to 4°
Line 23: Line 24:
* 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
-
* hold the cells on ice
+
* spin down again, remove supernatent
-
* Aliquot 50 μl of cells into chilled eppendorfs
+
* resuspend the pellet in the remaining liquid, bring the total volume to 1200 μl with EPB
-
* Add 1 μl of DNA for transformation
+
* Freeze 400 μl aliquots at -80 indefinitely or use immediately
-
* Mix and transfer to a chilled 1 mm gap electroporation cuvette
+
* Add 16 μl of transposome or plasmid DNA for transformation
-
* Pulse the cuvette at 1.75 KV twice, with a 5 second delay
+
* Mix and transfer to four chilled 1 mm gap electroporation cuvettes
-
* Add 200 μl of chilled 1161 medium from a 2 ml tube, mix with the pipet
+
* Pulse the cuvette at 1.5 KV
-
* Transfer the cuvette contents back into the same tube
+
* Add ml of chilled 1161 medium immediately, mix with the pipet, cover
-
** transfer may be easier with a gel loading tip to reach into the gap
+
* Place the cuvettes into the 30° incubator for outgrowth for 50 minutes
-
* 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
-
* Plate 100 μl aliquots on selective plates
+
** 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.
-
* Transfer 100 μl aliquots into 10 ml of selective medium
+
* Plate 360 μl aliquots on three selective plates
-
* grow plates and medium at 30° for colonies or color change
+
* grow plates for 1.5 - 2 days at 30° for colonies
 +
 
 +
==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
[[category:Mesoplasma florum]][[category:protocol]]
[[category:Mesoplasma florum]][[category:protocol]]

Current revision

back to protocols

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 (93.1 g/l)
  • chilled 1 mm electroporation cuvettes
  • 10 ml chilled ATCC 1161 medium
  • Selective medium or plates
    • Tetracycline resistance from TetM is higher than 200 μg/ml
    • Untransformed cells grow poorly at 4 μg/ml
    • Tet selective plates are at 15 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 1200 μl with EPB
  • Freeze 400 μl aliquots at -80 indefinitely or use immediately
  • Add 16 μl of transposome or plasmid DNA for transformation
  • Mix and transfer to four chilled 1 mm gap electroporation cuvettes
  • Pulse the cuvette at 1.5 KV
  • Add ml of chilled 1161 medium immediately, mix with the pipet, cover
  • Place the cuvettes into the 30° incubator for outgrowth for 50 minutes
    • 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 360 μl aliquots on three selective plates
  • grow plates for 1.5 - 2 days at 30° for colonies

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