λ Purification of phage arms (by sucrose density gradient centrifugation) experiments
Unlike insertion vectors, replacement vectors contain a filler fragment in the middle of the genome, which must be removed in order to accommodate the foreign DNA fragments. This process, commonly referred to as "λ-arm preparation", involves restriction enzyme digestion of λDNA to separate the two arms from the filler fragments and subsequent purification of the arms. This experiment is based on the "Guide to Molecular Cloning, Third Edition", translated by Huang Peitang et al.
Operation method
Purification of λ phage arms (by sucrose density gradient centrifugation) experiments
Principle
Unlike insertion vectors, replacement vectors contain a filler fragment in the middle of the genome, which must be removed in order to accommodate the foreign DNA fragments. This process, commonly referred to as "λ-arm preparation", involves restriction enzyme digestion of the λDNA to separate the two arms from the filler fragments and subsequent purification of the arms.
Materials and Instruments
λ Phage DNA Move I. Materials For more product details, please visit Aladdin Scientific website.
EDTA Ethanol N-butanol Sodium acetate Sucrose Gel Sampling Buffer TE
Agarose gel Beckman SW28 rotor or equivalent Dialysis bag Needle for hypodermic injection Wide-mouth pipette tip Sucrose gradient Water bath
1. Buffers and solutions
EDTA ( 0.5 mol/L, pH 8.0)
Ethanol
MgCI2 (1 mol/L)
n-Butanol
Sodium acetate (3 mol/L, pH 5.2)
Sucrose Gel Sampling Buffer
TE ( pH 7.6 and pH 8.0)
2. Gel
Agarose gels (0.5% and 0.7%) with 0.5 XTBE containing 0.5 μg/ml ethidium bromide
Agarose gel (0.5%, 75 mm thick) with 0.5 XTBE containing 0.5 μg/ml ethidium bromide.
3. Nucleic acids and oligonucleotides
λ Phage DNA
4. Centrifuges and rotors
Beckman SW28 rotor or equivalent in clear centrifuge tube, 25 x 89 mm ( e.g. Beckman SW28 centrifuge tube or equivalent)
5. Specialized equipment
Dialysis bag, boiled (for DNA)
Needle for hypodermic injection (21 gauge)
Wide-bore tip (large-bore tip)
Sucrose gradient (Two sucrose solutions, one containing 10% sucrose and the other containing 40% sucrose, were prepared and dissolved in a buffer containing 1 mol/L NaCl, 20 mol/L Tris-Cl (pH 8.0), and 5 mol/L EDTA (pH 8.0). Both solutions were passed through a 0.22 μm nitrocellulose membrane for sterilization.)
Pre-set in a water bath at 42 °C and 68 °C
II. Methods
Preparation of sucrose gradient
1. Prepare one or more 38 cm ( 10%~40%, m/V) sucrose gradients in a clear ultracentrifuge tube and allow to stand at 4°C for 1~2 h until used (Step 4).
2. digest and analyze approximately 60 μg of λ phage DNA. after precipitation with standard ethanol, dissolve the DNA in TE (pH 7.6) at a concentration of 150 μg/ml. a 0.2 μg fraction is set aside to serve as an electrophoretic control (step 7).
3. Add 1 mol/L MgCl2 to a final concentration of 10 mmol/L and incubate at 42℃ for 1 h to revert the sticky ends of λ phage DNA. Then, 0.2 μg of the fraction was subjected to 0.7% agarose electrophoresis to determine the success of replication.
4. For λ-phage DNA that has been complexed and digested, do not sample more than 75 μg of λ-phage DNA in each gradient, and the volume should be 500 μl or less.
5. Centrifuge the gradient at 15°C, 120,000 g (26,000 r/min in a Beckman SW28 rotor) for 24 hours.
6. Collect 0.5 ml of component by puncturing the bottom of the centrifuge tube with a 21-gauge needle.
7. Take two 15 μl portions of the collected sample from each of the two tubes and add 35 μl of water. Add 8 μl of Sucrose Gel Sampling Buffer, heat one portion at 68°C for 5 min and leave the other portion untreated. Analyze all samples on a thicker 0.5% agarose gel and use the full-length λ phage DNA and the digested DNA components reserved in step 2 as molecular quality standards.
8. After gel photography, the fractions containing the recombinant vector arms are localized and combined.
9. Dialyze the combined samples in a 1000-fold excess of TE (pH 8.0) at 4°C for 12-16 h with at least one buffer change.
10. Dialyze the sample several times with n-butanol to reduce the sample volume to less than 3 ml.
11. Recover dialyzed DNA by standard ethanol precipitation.
12. Dissolve the DNA in TE (pH 7.6) at a concentration of 300-500 μg/ml.
13. Determine the concentration of DNA by spectrophotometry and its purity by 0.5% agarose gel electrophoresis. Dispense into 1~5 μg portions and store at -20℃.