Team:CU-Boulder/Notebook/Protocols

From 2014.igem.org

(Difference between revisions)
Line 12: Line 12:
===Day 1===
===Day 1===
#Add a fresh colony of infectable cells to 50mL LB in 125mL flask.  
#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
#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
#Incubate for 60-90 minutes, shaking
#Incubate for 60-90 minutes, shaking
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===Day 2===
===Day 2===
-
1. Spin culture at 4,000 x g for 10 minutes
+
#Spin culture at 4,000 x g for 10 minutes
-
2. Transfer supernatant to a fresh conical. Repeat spin on supernatant
+
#Transfer supernatant to a fresh conical. Repeat spin on supernatant
-
3. Transfer the upper 90% of supernatant to a new conical
+
#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
+
#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
+
#Incubate at 4°C for at least 60 minutes
-
6. Centrifuge at 12,000 x g for 10 minutes. Carefully decant
+
#Centrifuge at 12,000 x g for 10 minutes. Carefully decant
-
7. Spin again briefly
+
#Spin again briefly
-
8. Gently resuspend pellet in 1.6mL 1x TBS
+
#Gently resuspend pellet in 1.6mL 1x TBS
-
9. Aliquot 800ul into 2 microfuge tubes. Proceed with both tubes
+
#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
+
#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
+
#Add 160ul of 2.5M NaCl/20% PEG-8000 solution to each
-
12. Let sit at room temperature for 5 minutes
+
#Let sit at room temperature for 5 minutes
-
13. Spin at 1300 x g for 10 minutes
+
#Spin at 1300 x g for 10 minutes
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14. Decant the supernatant
+
#Decant the supernatant
-
15. Spin briefly. Remove supernatant with pipet
+
#Spin briefly. Remove supernatant with pipet
-
16. Resuspend pellet in 200ul 1x TBS.  
+
#Resuspend pellet in 200ul 1x TBS.  
-
a. If desired, combine contents of both tubes into one
+
##If desired, combine contents of both tubes into one
2.5M NaCl/20% PEG-8000 (5x)
2.5M NaCl/20% PEG-8000 (5x)

Revision as of 04:48, 28 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.
    1. 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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