Team:Newcastle/Project/Labwork/PhilsProtocols

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(→Professor Phil Aldridge's Lab Protocols)
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=Professor Phil Aldridge's Lab Protocols=
=Professor Phil Aldridge's Lab Protocols=
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===Transforming DNA "Phil Style"===
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# Switch on heat block in flow to “LOW”.
 +
# Go get cells out of -80˚C and leave on ice for 30 minutes.
 +
# Check to see if heat block is at approx 42-45˚C.
 +
# Add DNA 1-20 ul to cells after vortexing them.
 +
# Leave on ice for EXACTLY 30 mins.
 +
# Place tubes in heat block for EXACTLY 50 secs.
 +
# Transfer back to ice for 2 mins.
 +
# After 2 mins add 0.9 ml LB and incubate for 45-60 mins. At 37˚C.
 +
# Plate out 200 ul and 200ul of a 1:10 dilution and start praying.
 +
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<b>Preparation of cells</b>
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# Dilute a ON culture 1:200 into at least 200 ml LB
 +
# Grow to an OD600 between 0.1 and 0.2 (usually 2-3hrs)
 +
# Spin down cells
 +
# Resuspend in 40ml ice cold 0.1 M CaCl2 and leave on ice for 30 min.
 +
# Spin down cells and resuspend in 1 ml 0.1M CaCl2
 +
# Transfer cells to an 1.7ml tube and add 105 ul glycerol and make sure you get a homogeneous solution
 +
# Aliquot in 100 ul volumes into clean 1.7 ml tubes and shock freeze in liquid nitrogen
 +
# store at –80˚C until used up
 +
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Revision as of 23:40, 7 July 2009

Professor Phil Aldridge's Lab Protocols

Transforming DNA "Phil Style"

  1. Switch on heat block in flow to “LOW”.
  2. Go get cells out of -80˚C and leave on ice for 30 minutes.
  3. Check to see if heat block is at approx 42-45˚C.
  4. Add DNA 1-20 ul to cells after vortexing them.
  5. Leave on ice for EXACTLY 30 mins.
  6. Place tubes in heat block for EXACTLY 50 secs.
  7. Transfer back to ice for 2 mins.
  8. After 2 mins add 0.9 ml LB and incubate for 45-60 mins. At 37˚C.
  9. Plate out 200 ul and 200ul of a 1:10 dilution and start praying.

Preparation of cells

  1. Dilute a ON culture 1:200 into at least 200 ml LB
  2. Grow to an OD600 between 0.1 and 0.2 (usually 2-3hrs)
  3. Spin down cells
  4. Resuspend in 40ml ice cold 0.1 M CaCl2 and leave on ice for 30 min.
  5. Spin down cells and resuspend in 1 ml 0.1M CaCl2
  6. Transfer cells to an 1.7ml tube and add 105 ul glycerol and make sure you get a homogeneous solution
  7. Aliquot in 100 ul volumes into clean 1.7 ml tubes and shock freeze in liquid nitrogen
  8. store at –80˚C until used up



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