Introduction

Alkaline phosphatase treatment is used to suppress the background of non-recombinants while performing restriction digestion with insertion vectors containing a single cloning site or polyclonal site using a single restriction enzyme. In this method, ligated DNA is first subjected to phenol: chloroform extraction and standard ethanol precipitation, respectively. It is then treated with restriction enzymes. The digested vector DNA is exposed to alkaline phosphatase treatment. The resulting reaction mixture is then mixed with SDS, EDTA, and proteinase K. The dephosphorylated DNA is stored in TE. 

This method is used to prevent self-ligation and reduce the background of non-recombinant phages.

 

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

Viral and Bacterial Strains

  • Commercially available bacteriophage λ packaging mixtures

Other Equipment

 

Method
  1. Mix 55µg of an appropriate bacteriophage vector DNA in λ annealing buffer to get a final volume of 150µl. Now incubate the DNA at 42oC for 60 minutes. This step allows the ends of phage DNA having ‘cos sites’ to anneal. 
  2. Prepare the reaction mixture by adding 20µl of 10X ligase buffer, 20µl of 10mM ATP (if required), and 0.2 to 0.5 Weiss units bacteriophage T4 DNA ligase per µg of phage DNA. Place this mixture at 16oC for 60 to 120 minutes. 
  3. Now perform phenol: chloroform extraction to extract this ligation mixture. The DNA forms closed circles and concatemers and are prone to shearing. Therefore, handle it carefully and do not vortex for extraction. Gently invert the tube to draw out emulsion formation during extraction.
  4. Centrifuge the mixture at room temperature for 1 minute to separate the organic and aqueous phases. Transfer the aqueous phase containing phage DNA to a new tube using an automated pipette equipped with wide-bore tips.
  5. Now carry out standard ethanol precipitation to recover DNA. Wash the pellet with 70% ethanol (1ml) and centrifuge again for 120 seconds. Remove the supernatant and place the open tube on the benchtop to facilitate the remaining ethanol evaporation. Resuspend and dissolve the damp pellet (containing DNA) in 150µl of TE (pH 8.0).
  6. Check the efficiency of ligation of cos termini of vector DNA by removing a 0.2µg aliquot and heat it in TE for five minutes at 68oC. Now cool it on ice and proceed for 0.6% agarose gel electrophoresis immediately. Use the following as controls.
    1. Heated non-ligated bacteriophage DNA
    2. Ligated, unheated bacteriophage DNA.
  7. Carry out DNA digestion using the desired number of restriction enzymes (according to previous protocols), extract the reaction mixture using phenol: chloroform, and then centrifuged to obtain vector DNA (as described in steps 3 and 4).
  8. Add 3M sodium acetate (0.1 volumes) and ethanol (2 volumes) to the reaction and centrifuge it at 4oC for 10 minutes to recover DNA. Wash the pellet with 1ml of 70% ethanol and centrifuge for 2 minutes again. Discard the supernatant containing ethanol and place the open tube on the bench to facilitate the remaining ethanol evaporation.
  9. Now add this precipitated DNA in 10mM Tris-Cl at a concentration of 100µg/ml. Store a 0.2µg aliquot on ice and treat the remaining DNA with an excess of CIP at 37oC for 60 minutes. For this, mix 0.01 units CIP and 0.1 volumes of 10X dephosphorylation buffer per 10µg of DNA. Then incubate this reaction mixture at 37oC for half an hour. Add another aliquot of CIP and set for another 30 minutes at 55oC.
  10. Add 0.5% SDS and 5mM EDTA respectively. Gently vortex to mix the solution and add proteinase K (to digest CIP completely) to a final concentration of 100µg/ml. Place the reaction mixture at 56oC for 30 minutes.
  11. Let the reaction mixture cool down to room temperature.
  12. Extract once with phenol: chloroform and once with chloroform to purify the DNA. Use standard ethanol precipitation to recover the DNA in the presence of sodium acetate (0.3M and pH 7.0).
  13. Ligate 0.2µg of the digested vector before and after CIP treatment to check the dephosphorylation efficiency. Package the DNA into phage particles and measure infectivity. This procedure reduces ligation and packaging efficiency by two to three orders of magnitude.

 

Precautions
  • The cohesive termini of DNA must be reannealed and ligated together before alkaline phosphatase treatment. In this way, vector arms’ ability to form concatamers with target DNA is improved, and the packaging efficiency is enhanced. 
  • Handle the ligated DNA carefully and mix gently. Avoid vortexing.
  • For blunt and recessed 5’ termini, carry the second half of incubation at 55oC. Blunt and recessed 5′ termini are poor substrates for CIP. However, dephosphorylation efficiency is increased at this temperature as DNA molecules are free to breathe and fray, allowing the enzymes to reach 5′ termini. 
  • For succeeding ligation reactions to work, proteinase K (used to digest CIP) must be completely removed after step 10. The proteinase K removal can be done by heating the reaction for 60 minutes in the presence of 5mM EDTA at 65oC and then extracting once with phenol: chloroform. 
  • Do not add ATP if the ligation buffer already contains ATP. 

 

Summary
  • Alkaline phosphatase treatment of vector DNA is used to reduce the background of non-recombinant phages and prohibit self-ligation. 
  • The DNA is ligated, extracted, and then subjected to restriction digestion. The digested vector DNA is treated with alkaline phosphatase and later on with Proteinase K, SDS, and EDTA. Phenol: chloroform extraction is done once again and then package DNA into phage particles. 

 

References
  1. Sambrook, J., & Russell, D. W. (2006). Alkaline Phosphatase Treatment of Bacteriophage λ Vector DNA. Cold Spring Harbor Protocols, 2006(1), pdb-prot3976.