Magni:Protocols/Transformation

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(New page: {{Magni}} <div style="padding: 10px; width: 720px; border: 5px solid #000000;"> = Transformation = *Thaw a vial of competent cells in ice (100 ul for each transformation). *Aliquot 100 ul...)
(Transformation)
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*Add 1 ml of sterile, pre-warmed LB and incubate the vial at 37degC, 220rpm for 1 h.
*Add 1 ml of sterile, pre-warmed LB and incubate the vial at 37degC, 220rpm for 1 h.
*Centrifuge the cells at 10000 rpm, 1 min in a microcentrifuge.
*Centrifuge the cells at 10000 rpm, 1 min in a microcentrifuge.
-
*Remove about 900 ul of supernatant and resuspend the pellet in the remaining liquid, thus concentrating the cells.
+
*Remove about 1 ml of supernatant and resuspend the pellet in the remaining liquid, thus concentrating the cells.
*Pipet the resuspended pellet on the pre-warmed LB agar plate and spread evenly.
*Pipet the resuspended pellet on the pre-warmed LB agar plate and spread evenly.
*Incubate at 37degC overnight or until colonies appear.
*Incubate at 37degC overnight or until colonies appear.

Revision as of 05:21, 13 November 2012

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Transformation

  • Thaw a vial of competent cells in ice (100 ul for each transformation).
  • Aliquot 100 ul of cells into a 1.5-ml tube.
  • Pre-warm a selective LB agar plate and a tube with sterile LB at 37degC.
  • Pre-heat the water bath at 42degC.
  • Add 1-10 ul of ligation (or 1 ul of miniprepped DNA, or 2-5 ul of resuspended DNA from iGEM plates) to 100 ul of cells.
  • Parafilm the vial and incubate in ice for 30 min.
  • Heat shock for 1 min.
  • Put the vial in ice for 1-2 min.
  • Add 1 ml of sterile, pre-warmed LB and incubate the vial at 37degC, 220rpm for 1 h.
  • Centrifuge the cells at 10000 rpm, 1 min in a microcentrifuge.
  • Remove about 1 ml of supernatant and resuspend the pellet in the remaining liquid, thus concentrating the cells.
  • Pipet the resuspended pellet on the pre-warmed LB agar plate and spread evenly.
  • Incubate at 37degC overnight or until colonies appear.

NOTES

  • When you don't have enough competent cells, their volume can be decreased from 100 ul to 50 ul.
  • When in a hurry, incubation times can be decreased: 5-10 min in ice instead of 30 min; 40 min in LB instead of 1 h.
  • When the water bath is not available, fill a becker with hot tap water, put a thermometer in the water and adjust the temperature to 42degC with cold/hot water.
  • When in a hurry and using Ampicillin plates, the 1-h incubation in LB can be avoided. Cells mix with DNA can be directly spread on a pre-warmed LB agar plate.
  • Heat-sensitive plasmids must be incubated at a proper temperature during the 1-h incubation in LB.
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