Team:CU-Boulder/Notebook/Protocols

From 2014.igem.org

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===Day 1===
===Day 1===
-
1.Add a fresh colony of infectable cells to 50mL LB in 125mL flask.  
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#Add a fresh colony of infectable cells to 50mL LB in 125mL flask.  
a.Include phagemid antibiotic only.  
a.Include phagemid antibiotic only.  
b.Grow at 37°C, 250rpm until OD is between 0.03 and 0.05
b.Grow at 37°C, 250rpm until OD is between 0.03 and 0.05
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2.Add the helper phage to a final concentration of 1 x10^8 phage/mL
+
#Add the helper phage to a final concentration of 1 x10^8 phage/mL
-
3.Incubate for 60-90 minutes, shaking
+
#Incubate for 60-90 minutes, shaking
-
4.Add Helper Phagemid antibiotic to a high concentration
+
#Add Helper Phagemid antibiotic to a high concentration
-
5.Grow for 14-18 hours at 37°C, shaking
+
#Grow for 14-18 hours at 37°C, shaking
===Day 2===
===Day 2===

Revision as of 18:48, 26 September 2014


Contents

Amplification of Phage using Helper Phage

Need

  • Plate of infectable cells that contain F’ episome
  • 2.5M NaCl/20% PEG-8000
  • 1x TBS

Day 1

  1. Add a fresh colony of infectable cells to 50mL LB in 125mL flask.

a.Include phagemid antibiotic only. b.Grow at 37°C, 250rpm until OD is between 0.03 and 0.05

  1. Add the helper phage to a final concentration of 1 x10^8 phage/mL
  2. Incubate for 60-90 minutes, shaking
  3. Add Helper Phagemid antibiotic to a high concentration
  4. Grow for 14-18 hours at 37°C, shaking

Day 2

1. Spin culture at 4,000 x g for 10 minutes 2. Transfer supernatant to a fresh conical. Repeat spin on supernatant 3. Transfer the upper 90% of supernatant to a new conical 4. Add 0.2 volume of 2.5M NaCl/20% PEG-8000 to the new conical. Gently mix 5. Incubate at 4°C for at least 60 minutes 6. Centrifuge at 12,000 x g for 10 minutes. Carefully decant 7. Spin again briefly 8. Gently resuspend pellet in 1.6mL 1x TBS 9. Aliquot 800ul into 2 microfuge tubes. Proceed with both tubes 10. Spin sample for 1 minute to pellet remaining cells. Transfer supernatant to fresh tubes 11. Add 160ul of 2.5M NaCl/20% PEG-8000 solution to each 12. Let sit at room temperature for 5 minutes 13. Spin at 1300 x g for 10 minutes 14. Decant the supernatant 15. Spin briefly. Remove supernatant with pipet 16. Resuspend pellet in 200ul 1x TBS. a. If desired, combine contents of both tubes into one

2.5M NaCl/20% PEG-8000 (5x) PEG-8000 100 g NaCl 75 g H2O 400 mL Bring final volume to 500 mL

TBS (1x) Tris 6.05 g NaCl 8.76 g H2O 800 mL Adjust pH to 7.6 with 1M HCl Adjust volume to 1 L



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