Introduction

At each end of the central stuffer fragment, a series of restriction sites arranged in opposite directions are present in replacement vectors. For instance, in EMBL3A, the order of restriction site in the left polyclonal site is SalI-BamHI-EcoRI, and that of the right polyclonal site is EcoRI-BamHI-SalI. Digestion of this type of vector with EcoRI and BamHI generates left and right arms with BamHI termini, a stuffer fragment with EcoRI termini, and short fragments of polyclonal sites having BamHI and EcoRI termini. Spun column centrifugation and differential precipitation with isopropanol can easily remove these segments.

The arms can proficiently ligate to target DNAs that carry BamHI compatible termini but cannot ligate with EcoRI termini of the stuffer fragment. This suppression of ligation to stuffer fragments can help to reduce the number of non-recombinant phages. The proportion of non-recombinant phages in cDNA and genome libraries can be reduced by ‘two order of magnitude’ in this way. When used in combination with ‘genetic selection,’ this method can decrease the number of non-recombinant phages by ‘hundredfold.’ Therefore, with numerous replacement vectors, the following are not required:

• Removal of stuffer fragment
• Purification of arms using gel electrophoresis or sucrose density centrifugation

Materials

Enzymes and Solutions

• • Chloroform
  • Ethanol 
  • 0.5M EDTA (pH 8.0)
  • 10% w/v SDS
  • Phenol: Chloroform (1:1 v/v)
  • 3M Sodium Acetate  (pH 5.2 and 7.0)
  • 10mM Tris-chloride (pH 8.3)
  • ATP (10mM)
  • Proteinase K 
  • Bacteriophage T4 DNA ligase
  • Restriction Endonucleases
  • Cell intestinal alkaline phosphatase (CIP)
  • Bacteriophage λ DNA

Buffers 

• TE (pH 7.6 and 8.0)
• λ Annealing Buffer
  •  10mM MgCl2
  • 100mM Tris-chloride
• 10X Dephosphorylation Buffer
  • 10mM MgCl2 
  • 10mM ZnCl2  
  • 100mM Tris-chloride

Viral and Bacterial Strains

• Commercially available bacteriophage λ packaging mixtures

Other Equipment

• Micropipette 

• Micropipette tips 

• Eppendorf

• Water Bath  (Preset to 16oC, 42oC, 56oC and 68oC)

Method

1. Dissolve 25 to 50µg of bacteriophage DNA λ (purified from a replacement vector) in TE to achieve a final volume of 170µl. 
2. Take two appropriate 10X restriction enzyme buffers. Add 20µl of one of them into the above mixture. Now take out two aliquots (to be used as controls during gel electrophoresis and ligation), each having 0.2µg of undigested bacteriophage DNA. Store these aliquots on ice.
3. Add 100-200 units (fourfold excess) of one of the two restriction enzymes to the digestion mixture and place it on ice for 4 hours at the recommended temperature.
4. Cool this digestion mixture to 0oC. Again remove two aliquots, each of 0.2µg. Incubate one of these aliquots and one of the aliquots from step 2 at 68oC for 10 minutes to disrupt the phage genome’s cohesive termini. Add approximately 10µl of sucrose gel loading buffer to the samples and immediately perform 0.7% agarose gel electrophoresis for the samples. If the digestion is complete, no DNA will migrate to the position of undigested control bands. Preferably two or three smaller DNA fragments will be seen. The number of these fragments depends on the number of cleavage sites present in the vector. 
5. Perform phenol: chloroform extraction twice and chloroform extraction once to purify the DNA.
6. Perform standard ethanol precipitation to recover DNA.
7.  Remix DNA in TE (pH 8.0) to get a final concentration of 250µg/ml. Add an appropriate 10X restriction enzyme buffer and digest the phage DNA with a second restriction enzyme. Use four-fold excess of the second restriction enzyme and incubate the digestion mixture for 4 hours at a temperature suggested by the manufacturer. 
8. Extract again twice with phenol: chloroform and once with chloroform. Use standard ethanol precipitation to recover DNA. Spun column centrifugation and differential precipitation with isopropanol are used to remove shorter fragments carrying polyclonal sites.
9. Again, dissolve digested DNA in TE (now at pH 7.6) to a final concentration of 400µg/ml (+/- 100µ/ml). Store an aliquot (0.2µg) at -20oC. 
10. Perform trial ligation reactions with 0.2µg of the vector digested with only the first restriction enzyme and 0.2 µg of the final product (from steps 4 and 9, respectively) to assess this DNA digestion procedure’s efficiency. Package equal amount, i.e., 0.1µg of phage DNA from each ligation mixture and determine the resulting bacteriophage particles’ titer. Follow a series of steps to achieve this.
11. Make the volumes of two DNA solutions with water to 17µl.
12. To each sample, add 2µl of 10X ligation buffer and 10mM ATP (if required).
13. Remove 10µl aliquots of each of the mixtures and store them on ice. 
14. Now add 0.2-0.5 Weiss units of T4 bacteriophage DNA ligase to the remaining mixtures and incubate these ligation mixtures at 16oC for 2 hours.
15. Package 0.1µg of the ligated and unligated samples and 0.1µg of undigested vector DNA, using a commercial packaging kit. Determine the titer of each packaged reaction.  

The packaging efficiency of the vector digested with one restriction enzyme increases by ‘three orders’ of magnitude after ligation and should be approximately 10% than that of undigested DNA. The efficiency of vector DNA digested with two restriction enzymes must be ‘two to three orders of magnitude less than that of vector DNA digested with a single restriction enzyme.

Checking the effectiveness of DNA Digestion

The effectiveness of restriction enzyme digestion determines the quality of the gene library and the efficiency of cloning target DNAs. You can determine the efficacy of the DNA digestion procedure by comparing the packaging ability of digested and undigested ligation mixtures described in step 10 above. One can also use a few other biochemical methods to measure the effectiveness of digestion.

1. End-label a small fraction of the first digestion product and then use this radiolabel to monitor restriction digestion with the second enzyme. Ligate the cohesive termini of vector DNA before digestion with the first restriction enzyme to prevent the excess radiolabel’s incorporation into the cohesive termini. Now, add 0.01-0.1µg of this radiolabelled DNA to the vector preparation. Second, restriction enzyme digestion causes radiolabel’s quantitative movement from large stuffer fragment (and arms) to small polyclonal site. Perform gel electrophoresis/ autoradiography/ phosphorimaging to monitor this movement.  
2. Perform PCR using a set of primers that lie to the right and left side of restriction sites. PCR products will be greatly reduced or eliminated by the cleavage of restriction sites. Compare the number of amplified PCR products having digested and undigested vector DNA to check digestion efficiency.   

However, these protocols do not directly measure cleaved DNA’s ability to perform as a cloning vector and are thus less recommended. 

Precautions

• Repeat the digestion and analysis if the number of non-recombinant phages is too high, i.e., greater than 104pfu/µg of cleaved vector DNA. 
• Carefully examine the number and yield of smaller fragments in step 4 and do not confuse them with partial digestion products.
• As far as ligation buffer is concerned, check for the presence of ATP. Some ligation buffers already have ATP. For such buffers, the addition of ATP is not required. 
• Sometimes, the background of plaques obtained after cleavage at a single restriction is unacceptably high. This high occurrence of plaques is due to a large number of mutants (resistant to the restriction enzyme) in the original population. Plaque-purify the phage λ vector frequently to overcome this problem. 
• Do not forget to remove control aliquots.

Summary

• Replacement vectors have a series of restriction sites oriented in opposite directions at each end of the central stuffer fragment. 
• The phage DNA is mixed with TE and treated with one restriction enzyme. The mixture treated with the first enzyme is then subjected to restriction digestion with the second enzyme.
• We can test the efficiency of the digestion process by analyzing the ligation mixtures of digested and undigested samples. The effectiveness of DNA digestion can also be tested by end-labeling or PCR.

References

1. Sambrook, J., & Russell, D. W. (2006). Preparation of Bacteriophage λ DNA Cleaved with Two Restriction Enzymes for Use as a Cloning Vector. Cold Spring Harbor Protocols, 2006(1), pdb-prot3987.