Team:CU-Boulder/Notebook/Phage Team

From 2014.igem.org

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__FORCETOC__
 +
 
 +
 
 +
==Week 1==
 +
 
 +
::'''Notes:''' Unless stated otherwise, all gels contain 2-log ladder
 +
 
 +
'''5/9'''
 +
*•Obtained BW23115 KanR cells- BW23115 cells that had their native CRISPR-Cas system knocked out by the insertion of a Kanamycin resistance gene
 +
**-Will also be called BW23115 or BW
 +
**-Conjugated BW23115 KanR cells with contain F’ notation (ex. BWF’)
 +
*•Obtained ER2738 cells that contain the F’ episome (no changes from NEB sample)
 +
**-Will also be called ER. Assume that all ER samples contain the F’ episome
 +
**-Streaked sample onto LB+Tet (20ug/mL) to select for colonies containing F’ episome
 +
 
 +
'''5/10'''
 +
*•Did receive colonies from 5/9 selection
 +
 
 +
==Week 2==
 +
 
 +
'''5/12'''
 +
*•Need to conjugate BW23115 KanR cells with the F’ episome
 +
**-Set up overnight cultures of ER2738 and BW23115 KanR
 +
**-When mixed, ER2738 will donate it’s F’ episome and BW23115 KanR will receive the F’ episome. F’ episome confers Tetracycline resistance
 +
 
 +
'''5/13'''
 +
*•Started M13 Amplification: Amplify M13 phage using the M13K07 Helper Phage
 +
**-Let precipitated in NaCl/PEG solution overnight
 +
**-Possible sources of error
 +
::::Did not sterilize 2.5M NaCl/20% PEG-8000 solution
 +
::::Added 4-fold PEG solution
 +
****•Compensated by adding more LB
 +
::::During precipitation, put sample in -20C for 30 minutes before realizing mistake and moving to it to 4C. Sample partially froze
 +
*•Conjugated BW23115 with F’ episome
 +
**-Added 1mL BW23115 to 1mL ER2738 overnight culture
 +
**-Incubated at 37C for 30 minutes, shaking
 +
**-Plated on LB+Kan(50ug/mL)+Tet(20ug/mL)
 +
::::To select for BW cells that took the F’ episome (containing Tet resistance)
 +
 
 +
'''5/14'''
 +
*•Finished the M13 Amplification
 +
**-Visualized product on UV-vis. There was a tall spike at 269nm indicating that DNA was present. Did not test at 320nm.
 +
*•Results of BW23115 Conjugation
 +
**-Many colonies indicating successful conjugation of F’ episome into BW23115
 +
**-Set up overnight to make freeze down tomorrow
 +
*•Set up overnight of ER2738 to make chemically competent tomorrow
 +
 
 +
'''5/15'''
 +
*•Made freeze down of BW23115 KanR F’
 +
**-BW23115 E. coli strain with Kanamycin resistance gene inserted into genome and with F’ episome
 +
*•Made chemically competent ER2738 cells
 +
*•Transformation of Litmus28i (from NEB) into chemically competent ER2738 cells
 +
**-Added 1ul Litmus28i plasmid to 40ul competent cells
 +
**-Plated on LB + Amp(100ug/mL)
 +
**-Purpose: To make M13 phage that package Litmus28i DNA. Need phagemid (Litmus28i) DNA in infectable cells (cells containing F’ episome) to introduce M13K07 Helper Phage and make phage.
 +
 
 +
'''5/16'''
 +
*•Results of 5/15 transformation
 +
**-No growth for No DNA control
 +
**-Many colonies for sample
 +
 
 +
 
 +
==Week 3==
 +
 
 +
'''5/19'''
 +
*•M13 Amplification to isolate M13-Litmus28i phage
 +
**-Cells: ER2738 cells containing Litmus28i phagemid
 +
**-Helper Phage: M13K07
 +
**-Not much phage was precipitated
 +
*•Set up overnight culture of ER2738 to infect tomorrow
 +
 
 +
'''5/20'''
 +
*•Infected ER2738 cells with M13-Litmus28i phage
 +
**-Plated only on Ampicillin(100ug/mL)  (should have also plated on kanamycin)
 +
**-Infected for 4-5 hours-> should have only infected for 30 minutes maximum. This extra time gives the cells that were infected with M13-M13K07 the time to produced M13-M13K07 phage and reinfect
 +
 
 +
'''5/21'''
 +
*•Results from M13-Litmus28i infection of ER2738
 +
**-Solid lawn of growth for diluted and non-diluted
 +
**-Also sickly looking growth
 +
*•Set up overnights
 +
**-ER2738 cells containing Litmus28i for freeze down
 +
**-BW23115 with F’ episome to make chemically competent cells
 +
**-ER2738 to redo infection
 +
 
 +
'''5/22'''
 +
*•Tested absorbance of phage produced through M13 amplification on 5/19
 +
**-Low absorbance of 0.018 at 269nm but no detection at wavelength 320nm
 +
**-Decided to redo M13 amplification
 +
*•Made chemically competent BW23115 with f-episome
 +
*•Made freeze down of ER2738 containing Litmus28i
 +
*•Set up overnight of ER2738 containing Litmus28i to redo M13 amplification tomorrow
 +
 
 +
'''5/23'''
 +
*•Protocol switch to make phage using phagemid
 +
**-“M13 Amplification” protocol should only be used to make more M13-M13K07, not to make M13 phage containing a different phagemid
 +
**-Switched to new protocol (“Use of M13K07 Helper Phage for isolation of single stranded phagemid DNA” by NEB. Made modifications (see our protocols) to isolate phage rather than single-stranded DNA)
 +
**-Making phage….
 +
 
 +
 
 +
 
 +
 
 +
 
 +
*•Made fresh antibiotics
 +
**-Chloramphenicol (34 ng/mL)
 +
::::1.44g chloramphenicol into 42mL EtOH
 +
**-Ampicillin (50 ng/mL)
 +
::::4g ampicillin into 80mL mili-Q H2O

Revision as of 05:32, 4 October 2014


Contents

Week 1

Notes: Unless stated otherwise, all gels contain 2-log ladder

5/9

  • •Obtained BW23115 KanR cells- BW23115 cells that had their native CRISPR-Cas system knocked out by the insertion of a Kanamycin resistance gene
    • -Will also be called BW23115 or BW
    • -Conjugated BW23115 KanR cells with contain F’ notation (ex. BWF’)
  • •Obtained ER2738 cells that contain the F’ episome (no changes from NEB sample)
    • -Will also be called ER. Assume that all ER samples contain the F’ episome
    • -Streaked sample onto LB+Tet (20ug/mL) to select for colonies containing F’ episome

5/10

  • •Did receive colonies from 5/9 selection

Week 2

5/12

  • •Need to conjugate BW23115 KanR cells with the F’ episome
    • -Set up overnight cultures of ER2738 and BW23115 KanR
    • -When mixed, ER2738 will donate it’s F’ episome and BW23115 KanR will receive the F’ episome. F’ episome confers Tetracycline resistance

5/13

  • •Started M13 Amplification: Amplify M13 phage using the M13K07 Helper Phage
    • -Let precipitated in NaCl/PEG solution overnight
    • -Possible sources of error
Did not sterilize 2.5M NaCl/20% PEG-8000 solution
Added 4-fold PEG solution
        • •Compensated by adding more LB
During precipitation, put sample in -20C for 30 minutes before realizing mistake and moving to it to 4C. Sample partially froze
  • •Conjugated BW23115 with F’ episome
    • -Added 1mL BW23115 to 1mL ER2738 overnight culture
    • -Incubated at 37C for 30 minutes, shaking
    • -Plated on LB+Kan(50ug/mL)+Tet(20ug/mL)
To select for BW cells that took the F’ episome (containing Tet resistance)

5/14

  • •Finished the M13 Amplification
    • -Visualized product on UV-vis. There was a tall spike at 269nm indicating that DNA was present. Did not test at 320nm.
  • •Results of BW23115 Conjugation
    • -Many colonies indicating successful conjugation of F’ episome into BW23115
    • -Set up overnight to make freeze down tomorrow
  • •Set up overnight of ER2738 to make chemically competent tomorrow

5/15

  • •Made freeze down of BW23115 KanR F’
    • -BW23115 E. coli strain with Kanamycin resistance gene inserted into genome and with F’ episome
  • •Made chemically competent ER2738 cells
  • •Transformation of Litmus28i (from NEB) into chemically competent ER2738 cells
    • -Added 1ul Litmus28i plasmid to 40ul competent cells
    • -Plated on LB + Amp(100ug/mL)
    • -Purpose: To make M13 phage that package Litmus28i DNA. Need phagemid (Litmus28i) DNA in infectable cells (cells containing F’ episome) to introduce M13K07 Helper Phage and make phage.

5/16

  • •Results of 5/15 transformation
    • -No growth for No DNA control
    • -Many colonies for sample


Week 3

5/19

  • •M13 Amplification to isolate M13-Litmus28i phage
    • -Cells: ER2738 cells containing Litmus28i phagemid
    • -Helper Phage: M13K07
    • -Not much phage was precipitated
  • •Set up overnight culture of ER2738 to infect tomorrow

5/20

  • •Infected ER2738 cells with M13-Litmus28i phage
    • -Plated only on Ampicillin(100ug/mL) (should have also plated on kanamycin)
    • -Infected for 4-5 hours-> should have only infected for 30 minutes maximum. This extra time gives the cells that were infected with M13-M13K07 the time to produced M13-M13K07 phage and reinfect

5/21

  • •Results from M13-Litmus28i infection of ER2738
    • -Solid lawn of growth for diluted and non-diluted
    • -Also sickly looking growth
  • •Set up overnights
    • -ER2738 cells containing Litmus28i for freeze down
    • -BW23115 with F’ episome to make chemically competent cells
    • -ER2738 to redo infection

5/22

  • •Tested absorbance of phage produced through M13 amplification on 5/19
    • -Low absorbance of 0.018 at 269nm but no detection at wavelength 320nm
    • -Decided to redo M13 amplification
  • •Made chemically competent BW23115 with f-episome
  • •Made freeze down of ER2738 containing Litmus28i
  • •Set up overnight of ER2738 containing Litmus28i to redo M13 amplification tomorrow

5/23

  • •Protocol switch to make phage using phagemid
    • -“M13 Amplification” protocol should only be used to make more M13-M13K07, not to make M13 phage containing a different phagemid
    • -Switched to new protocol (“Use of M13K07 Helper Phage for isolation of single stranded phagemid DNA” by NEB. Made modifications (see our protocols) to isolate phage rather than single-stranded DNA)
    • -Making phage….



  • •Made fresh antibiotics
    • -Chloramphenicol (34 ng/mL)
1.44g chloramphenicol into 42mL EtOH
    • -Ampicillin (50 ng/mL)
4g ampicillin into 80mL mili-Q H2O
Retrieved from "http://2014.igem.org/Team:CU-Boulder/Notebook/Phage_Team"