Determination of DNA content in λ phage progeny and lysates by gel electrophoresis experiment
This program describes a method for rapid estimation of the DNA content in λ phage protospecies. The method can be used firstly to find out whether the yield of phage particles in protospecies and lysates can meet the needs of mass purification, and secondly to test the purification process to find the problematic steps. The source of this experiment is "Guide to Molecular Cloning Experiments, Third Edition", translated by Huang Peitang et al.
Operation method
Experiments on the determination of DNA content in λ phage progeny and lysates by gel electrophoresis
Principle
This program describes a method for rapid estimation of DNA content in λ phage protospecies. The method can be used firstly to find out whether the yield of phage particles in the original seed and lysates is sufficient for large quantities of purification, and secondly to test the purification process in order to find problematic steps.
Materials and Instruments
λ Phage DNA λ Phage lysate or protospecies Move I. Materials For more product details, please visit Aladdin Scientific website.
2.5X SDS-EDTA dye mixture DNase I dilution buffer Tryptic DNase I
Agarose gel Water bath
1. Buffers and solutions
2.5X SDS-EDTA dye mixture
2. enzyme and buffer
DNase I dilution buffer, tryptic DNase I (1 mg/ml)
3. gel
Agarose gel (0.7%) with 0.5X TBE containing 0.5 μg/ml ethidium bromide
4. nucleic acids and oligonucleotides
λ Phage DNA ( control DNA)
5. Specialized equipment
Water bath pre-set at 65°C
6. Carriers and strains
λ Phage lysates or proto-species
II. Methods
1. Configure the working solution of tryptic DNase Ⅰ (1 μg/ml) as follows: 1 μl of DNase Ⅰ reservoir solution was diluted with 1 ml of ice-cold Dnase Ⅰ dilution buffer.
2. Gently turn the sealed test tube up and down several times to mix the solution, taking care not to produce bubbles or foam. Store the working solution in an ice bath before use and discard after use.
A clear band of λ phage DNA is obtained in the crude plate lysate. However, liquid lysates generally contain bacterial DNA fragments that will interfere with the phage DNA band in agarose gels. This problem can be avoided by treating the phage pellet with tryptic DNase I before treating with SDS and EDTA to release the λDNA.
3. Transfer 10 μl of phage lysate or crude product of the original species to a microcentrifuge tube, add 1 μl of Tryptic DNase working solution and incubate at 37℃ for 30 min.
4. Add 4 μl of 2.5X SDS-EDTA dye mixture and incubate at 65 °C for 5 min.
This step ruptures the phage shell, releasing the viral DNA and inactivating the DNase.
5. Spread the sample onto a 0.7% agarose gel containing 0.5 μg/ml ethidium bromide.
Use 5 ng, 25 ng and 100 ng of λ phage DNA as control.
6. Electrophoresis was performed at an electric field of less than 5 V/cm until the bromophenol blue migrated 3-4 cm.
If the voltage is too high, the DNA bands will tail off.
7. Observe the gel under UV light. Use the fluorescence density of the standard DNA as a control to estimate the amount of λ phage DNA in the test sample.
10 μl of high titer lysate should contain 10-50 ng of phage DNA.