UA Biophysics:Protocols:Marking T4 DNA with DAPI: Difference between revisions
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==Recipe== | ==Recipe== | ||
For this procedure, a modified version of the methodology given by Furukawa et. al.[1] elaborated for a simple of 10^9 PFU/ μl will be used. E. coli B is cultured to 0.5 x 10^8 cells per ml in 25 ml of TSB 30 gr/l (OXOID). The phage is added ensuring a multiplicity of infection (MOI) of 5 particles per cell | For this procedure, a modified version of the methodology given by Furukawa et. al.[1] elaborated for a simple of 10^9 PFU/ μl will be used: | ||
1. E. coli B is cultured to 0.5 x 10^8 cells per ml in 25 ml of TSB 30 gr/l (OXOID). | |||
2. The phage is added ensuring a multiplicity of infection (MOI) of 5 particles per cell. | |||
3. Simultaneously, DAPI (4’,6-diamidino-2-phenylindle from SIGMA) is added to a concentration of 5 μg/ml. | |||
4. 6 hours later cloroform is added to 0.1%. | |||
==Other Links== | ==Other Links== | ||
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[1] Furukawa, H., Kuroiwa, T. & Mizushima, S. DNA injection during bacteriophage T4 infection of Escherichia coli. J. Bacteriol. 154, 938–945 (1983). | [1] Furukawa, H., Kuroiwa, T. & Mizushima, S. DNA injection during bacteriophage T4 infection of Escherichia coli. J. Bacteriol. 154, 938–945 (1983). | ||
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Latest revision as of 13:55, 8 September 2015
WARNING
This protocol CAN'T be done at the installations of the biophysics laboratory as it compromises the safety of the biological samples present at it.
Purpose
Mark T4 DNA with 4’,6-diamidino-2-phenylindle (DAPI).
Recipe
For this procedure, a modified version of the methodology given by Furukawa et. al.[1] elaborated for a simple of 10^9 PFU/ μl will be used:
1. E. coli B is cultured to 0.5 x 10^8 cells per ml in 25 ml of TSB 30 gr/l (OXOID).
2. The phage is added ensuring a multiplicity of infection (MOI) of 5 particles per cell.
3. Simultaneously, DAPI (4’,6-diamidino-2-phenylindle from SIGMA) is added to a concentration of 5 μg/ml.
4. 6 hours later cloroform is added to 0.1%.
Other Links
- Fluorescence Ex/Em spectra of DAPI bound to DNA: [1]
References
[1] Furukawa, H., Kuroiwa, T. & Mizushima, S. DNA injection during bacteriophage T4 infection of Escherichia coli. J. Bacteriol. 154, 938–945 (1983).
