Purification of Bacteriophage λ Arms using Sucrose Density Gradients

Introduction

Replacement vectors or substitution vectors are prepared by removing the central filler segment of phage DNA to accommodate the fragments of foreign DNA. This procedure is known as the “preparation of λ arms.” Restriction endonucleases are used to separate the two arms of phage DNA from the central stuffer fragment. The following methods are used to purify bacteriophage λ arms:

1. Sucrose density gradient based centrifugation
2. NaCl density gradient based centrifugation
3. 0.5% preparative agarose gel electrophoresis

 2µg/ml Ethidium Bromide can be added to NaCl or sucrose density gradients to visualize different DNA species within the gradient. With experience, the fractions of phage DNA containing annealed arms can be pooled without former examination by agarose gel electrophoresis. In some cases, for instance, when the digested DNA is to be treated with alkaline phosphatase, the preliminary ligation of cohesive termini before exposure to restriction endonucleases is preferred.

One can use the method described below to purify the arms of any bacteriophage λ vector.

Materials

Buffers, Gels, and Solutions

• 0.5M EDTA  (pH 8.0)
• Ethanol
• Tris-Cl
• 3M Sodium Acetate (pH 5.2)
• TE (pH 7.6 and 8.0)
• Sucrose Loading Buffer
• N-butanol
• 1M NaCl 
• 1M MgCl2  
• 0.5% and 0.7% agarose gel cast in 0.5X TBE, containing 0.5µg/ml EtBr
• 0.5% agarose gel (75mm thick) cast in 0.5X TBE, containing 0.5µg/ml EtBr

     Nucleic acids

• Bacteriophage λ DNA

     Other Equipment

• Z206A Compact Centrifuge 

• Clear Centrifuge Tubes 

• 21 Gauge Hypodermic Needles 

• Sterilized dialysis tubing   

• Wide bore tips 
• Water bath  (preset to 42oC and 68oC)

• Gel Electrophoresis Equipment 

Preparation of Sucrose Density Gradients

Prepare two sucrose solutions containing 10% w/v and 40% w/v sucrose respectively in NaCl (1M), Tris Chloride (20mM and pH 8.0), and 5mM EDTA. Sterilize the sucrose solutions by using 0.22µm thick nitrocellulose filters. 

Sucrose density gradient can also be linearly established by diffusion. This method is preferably used when large numbers of gradients are required. Prepare four sterilized sucrose solutions (10%, 20%, 30% and 40% w/v) in NaCl (1M), Tris Chloride (20mM and pH 8.0), and 5mM EDTA. To form a 38ml density gradient, layer 9.5ml of each sucrose solution one above another in the descending order of density, i.e., 40% w/v solution in the bottom and 10% on the top. Some researchers also oppositely prepare the gradient, i.e., by placing 10% sucrose solution in the bottom, overlaying 20%, 30%, and 40% sucrose solutions, respectively. 

Continuous density gradients can also be prepared in a gradient maker. Each gradient requires approximately 15 minutes to pour at room temperature. Each gradient can hold 60-75µg of DNA.

Method

• Prepare one or more sucrose density gradients as described above in clear centrifuge tubes and incubate them at 4oC for 60 to 120 minutes in a separate corner. 
• Dissolve approximately 60µg of DNA in TE and perform restriction digestion and analysis as described in the previous protocols. Perform standard ethanol precipitation and then dissolve DNA in TE at 150µg/ml. Remove 0.2µg aliquot to be used as a control in gel electrophoresis.
• Add MgCl2 to a concentration of 10mM and let the cohesive termini anneal by placing the mixture at 42oC for 60 minutes. To check whether annealing has occurred or not, separate 0.2µg aliquot of the mixture and analyze it through 0.7% agarose gel electrophoresis. Use 0.2µg aliquot of intact DNA and 0.2µg of annealed DNA (heated at 68oC for 10 minutes to melt cohesive termini) as markers.
• Load 500µl of 75µg annealed and digested phage DNA onto each gradient. Centrifuge the gradients at 26000rpm for 24 hours at 15oC. 
• Collect 0.5ml DNA fractions using a hypodermic needle at the bottom of the centrifuge tube.
• Separate out 15µl from every third fraction and mix it with 35µl water and 8µl of sucrose gel loading buffer. Make two aliquots from each fraction. Keep one aliquot untreated and heat the other at 68oC for 5 minutes. Analyze the samples using thick 0.5% gel electrophoresis. Use markers (mentioned above) along with the samples. To make the markers’ electrophoretic mobilities compared to the samples, adjust their sucrose and salt concentrations accordingly.

 The rate of migration of annealed arms is similar to that of the undigested DNA.

• Photograph the gel and then locate and pool the fractions of annealed arms.
• Perform the dialysis of pooled fractions against 1000X excess of TE (pH 8.0) at 4oC for 12 to 16 hours. Change the buffer at least once and allow a two to three-fold volume increase during dialysis. 
• Perform n-butanol extraction of the dialyzed sample several times to reduce the volume to less than 3ml.
• Perform standard ethanol precipitation to recover dialyzed DNA.
• Dissolve the extracted DNA in TE at a concentration of 400µg/ml (+/-100µg/ml).
• Measure the DNA concentration using a spectrophotometer such that 1.OD at 260nm is equal to 50µg/ml. Perform 0.5% agarose gel electrophoresis of one aliquot to analyze the purity of the sample. 
• Now store the DNA at -20oC in tubes of 1 to 5µg.

Precautions

• Be careful while loading DNA onto the gradient. A greater amount of DNA can overload the gradient, which results in poor separation of the filler segments from their arms.
• Make sure to use markers during gel electrophoresis. Adjust the salt and sucrose concentrations of these markers to make their ‘electrophoretic mobilities’ comparable.
• Do not use high voltages and buffers with high electrical resistance while running analytical gels, as overheating can result in the melting of cohesive termini. 
• Do not consider the visibly contaminated and undigested, unannealed DNA fractions (including both arms and central stuffer fragments) while photographing the gel. 
• If the pooled samples have a relatively smaller volume, do not dialyze the DNA. Instead, directly dilute it with TE to reduce the sucrose concentration to about 10%. Moreover, to remove the residual dye (EtBr), extract the purified phage arms two times with isoamyl alcohol. 

Applications

• We can use purified Bacteriophage λ Arms obtained by sucrose density gradient-based centrifugation for sub-cloning artificial chromosomes, such as yeast or bacteria chromosomes.

• Purified phage arms can also be used for the construction of genomic libraries and phage display systems. 

Strengths and Limitations

Sucrose density gradients have higher resolving power; therefore, phage arms obtained by this method have higher purity. However, this method is more time-consuming than the other gradients used for the purification of arms. 

Sometimes, DNA can be digested with a restriction enzyme that cleaves within the central filler segment and not the arm. This approach reduces the size of the stuffer fragment and improves the separation of filler segments from the arms, ultimately generating termini incompatible with that of the arms.

Purified arms of the popular vectors such as EMBL3, EMBL4, λgt10 and λgt11, etc., are available commercially. However, if the user is inexperienced or happens to make a small mistake, these arms become worthless. In some small scale cloning projects, such as when sub-cloning from an individual yeast artificial chromosome, the use of commercially available arms’ sources seem to be less expensive; however, if these arms are to be regularly used, such as for the construction of genomic libraries, it is preferred to use domestically available arms prepared by density gradient based centrifugation.

Summary

Sucrose density gradients have higher resolving power; therefore, phage arms obtained by this method have higher purity. However, this method is more time-consuming than the other gradients used for the purification of arms. 

Sometimes, DNA can be digested with a restriction enzyme that cleaves within the central filler segment and not the arm. This approach reduces the size of the stuffer fragment and improves the separation of filler segments from the arms, ultimately generating termini incompatible with that of the arms.

Purified arms of the popular vectors such as EMBL3, EMBL4, λgt10 and λgt11, etc., are available commercially. However, if the user is inexperienced or happens to make a small mistake, these arms become worthless. In some small scale cloning projects, such as when sub-cloning from an individual yeast artificial chromosome, the use of commercially available arms’ sources seems to be less expensive; however, if these arms are to be regularly used, such as for the construction of genomic libraries, it is preferred to use domestically available arms prepared by density gradient based centrifugation.

References

1. Sambrook, J., & Russell, D. W. (2006). Purification of Bacteriophage λ Arms: Centrifugation through Sucrose Density Gradients. Cold Spring Harbor Protocols, 2006(1), pdb-prot3990.