Extraction of λ phage DNA from large-scale cultures with formamide
Instead of SDS/proteinase K, formamide can be used to remove the shell from purified phage particles. This method is not very efficient, but in a sense it is faster than the previous method. The source of this experiment is "Guide to Molecular Cloning Experiments, Third Edition" translated by Huang Peitang et al.
Operation method
Experimental extraction of λ phage DNA from mass cultures with formamide
Principle
Instead of SDS/proteinase K, formamide can be used to remove the shell from purified phage particles. This method is not very efficient, but in a sense it is faster than previous manipulations.
Materials and Instruments
Restriction endonuclease λ Phage particles Move I. Materials For more product details, please visit Aladdin Scientific website.
EDTA Ethanol Formamide NaCl TE Tris-Cl
Agarose gel Borosilicate Pasteur pipette
1. Buffers and solutions
EDTA ( 0.5 mol/L, pH 8.0)
Ethanol
Formamide, deionized
NaCl ( 5 mol/L)
TE (pH 7.6 and pH 8.0)
Tris-Cl ( 2 mol/L, pH 8.5)
2. Enzyme and buffer
Restriction endonuclease
3. gels
Agarose gel (0.7%) with 0.5X TBE containing 0.5 μg/ml ethidium bromide
4. Specialized equipment
Borosilicate Pasteur pipettes (sealed) or Shepherd hooks
5. Carriers and strains
λ Phage particles
II. Methods
1. If necessary, remove CsCl from the phage particle sample as described in Steps 1 to 4 of Scheme 11.
2. Determine the volume of the phage particle sample.
3. Add 0.1 times the volume of 2 mol/L Tris (pH 8.5), 0.05 times the volume of 0.5 mol/L EDTA (pH 8.0), and 1 times the volume of deionized formamide, and incubate at 37°C for 30 min.
During the incubation, the milky suspension of phage particles turned into a clear solution.
4. Add 1 times the volume of H2O (equal to the final volume in Step 3) and 6 times the volume of ethanol (each times the final volume in Step 3).
5. Adhere the λ phage DNA precipitate to the end of a sealed borosilicate Pasteur pipette or Shepherd's hook and transfer to a microcentrifuge tube containing 70% ethanol.
6. Centrifuge briefly (10 s ) to collect the DNA precipitate.
Avoid prolonged centrifugation as the DNA will be too compact to be dissolved.
7. Remove the supernatant and leave the tube open on the bench for a few minutes to allow the ethanol to evaporate. Add 300 μl of TE and re-dissolve the moist DNA precipitate by tapping the walls of the centrifuge tube. Avoid using vortex shaking if possible.
8. Add 6 μl of 5 mol/L NaCl and 750 μl of ethanol to reprecipitate the DNA, collect the precipitate, and re-dissolve as described in steps 6 and 7.
9. Check the integrity of the phage DNA by analyzing the undigested or cut portion of the DNA (0.5 μg) with appropriate restriction enzymes by 0.7% agarose electrophoresis using appropriate size molecular mass standards.
10. Store the phage DNA at 4℃.