Molecular Techniques and Methods

Construction of cDNA Library into Lamda Zap Vector

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

Each organism and tissue type has a unique population of messenger RNA molecules. These mRNA populations are difficult to maintain, clone, and amplify; therefore, they must be converted to more stable DNA molecules (cDNA). Successful cDNA synthesis should yield full-length copies of the original population of mRNA molecules.

The quality of the cDNA library can be only as good as the quality of the mRNA. Pure, undegraded mRNA is essential for the construction of large, representative cDNA libraries.

Secondary structure of mRNA molecules can cause the synthesis of truncated cDNA fragments. In this case, treatment of the mRNA with a denaturant, such as methyl-mercuric hydroxide, prior to synthesis may be necessary.

Other potential difficulties include DNA molecules contaminating the mRNA sample. DNA can clone efficiently, and their introns can confuse results. RNase-free DNase treatment of the sample is recommended.

After synthesis, the cDNA is inserted into an Escherichia coli-based vector, and the library is screened for clones of interest. Since 1980, lambda has been the vector system of choice for cDNA cloning. The fundamental reasons are that in vitro packaging of lamda phage generally has a higher efficiency than plasmid transformation, and lamda libraries are easier to handle (amplify, plate, screen, and store) than plasmid libraries. However, most lamda vectors have the disadvantage of being poorer templates for DNA sequencing, site-specific mutagenesis, and restriction fragment shuffling, although this trend is reversing to some degree with the continued development of polymerase chain reaction (PCR) techniques.

The development of excisable lamda vectors, such as those based on restriction enzyme digestion, site-specific recombination, or filamentous phage replication, has increased the flexibility of DNA cloning. Lambda ZAP vector use an excision mechanism that is based on filamentous helper phage replication (e.g., M13).

In this procedure, a hybrid oligo(dT) linker-primer containing an XhoI site is used to make directional cDNA. This 48-base oligonucleotide was designed with a protective sequence to prevent the XhoI restriction enzyme recognition site from being damaged in subsequent steps and an 18-base poly(dT) sequence, which binds to the 3' poly(A) region of the mRNA template.

5'-GAGAGAGAGAGAGAGAGAGAGAGACTCGAGTTTTTTTTTTTTTTTTTTT-3'

First-strand synthesis is primed with the oligo(dT) linker-primer and is transcribed by reverse transcriptase in the presence of nucleotides and buffer. An RNaseH-deficient reverse transcriptase may produce larger yields of longer cDNA transcripts. The use of 5-methyl dCTP in the nucleotide mix during first-strand synthesis "hemi-methylates" the cDNA, protecting it from digestion during a subsequent restriction endonuclease reaction used to cleave the internal XhoI site in the linker-primer.

The cDNA/mRNA hybrid is treated with RNase H in the second-strand synthesis reaction. The mRNA is nicked to produce fragments that serve as primers for DNA polymerase I, synthesizing second-strand cDNA. The second-strand nucleotide mixture is supplemented with dCTP to dilute the 5-methyl dCTP, reducing the probability of methylating the second-strand, since the XhoI restriction site in the linker-primer must be susceptible to restriction enzyme digestion for subsequent ligation into the vector.

The uneven termini of the double-stranded cDNA must be polished with cloned Pfu DNA polymerase to allow efficient ligation of adapters. Adapters are complementary oligonucleotides which, when annealed, create a phosphorylated blunt end and a dephosphorylated cohesive end. This double-stranded adapter will ligate to other blunt termini on the cDNA fragments and to other adapters. Since the cohesive end is dephosphorylated, ligation to other cohesive ends is prevented. After the adapter ligation reaction is complete and the ligase has been inactivated, the molecules are phosphorylated to allow ligation to the dephosphorylated vector.

An XhoI digestion releases the adapter and protective sequence on the Oligo(dT) linker-primer from the 3'-end of the cDNA. These fragments are separated from the cDNA on a size fractionation column. The purified cDNA is then precipitated and ligated to the vector.




MATERIALS AND SOLUTIONS

10 x First-Strand Buffer (1 ml)
500 mM Tris-HCl (pH7.6) ------------------------ 500 ul of 1 M Tris-HCl
700 mM KCl -------------------------------------- 350 ul of 2 M KCl
100 mM MgCl2 ----------------------------------- 100 ul of 1 M MgCl2
Distilled H2O -------------------------------------- 50 ul


Methyl-Nucleotide Mixture (1 ml)
10 mM dATP ------------------------------------- 100 ul of 100 mM dATP
10 mM dGTP ------------------------------------- 100 ul of 100 mM dGTP
10 mM dTTP ------------------------------------- 100 ul of 100 mM dTTP
5 mM 5-methyl dCTP ----------------------------- 100 ul of 5-methyl dCTP
Distilled H2O -------------------------------------- 600 ul


Oligo (dT) Linker-Primer (XhoI) (3.0 ug at 1.5 ug/ul)
5'-GAGAGAGAGAGAGAGAGAGAGAGACTCGAGTTTTTTTTTTTTTTTTTTT-3'


10 x Second-Strand Buffer (1 ml)
700 mM Tris-HCl (pH 7.4) ------------------------ 700 ul of 1 M Tris-HCl
100 mM (NH4)2SO4 ------------------------------ 100 ul of 1 M (NH4)2SO4
50 mM MgCl2 ------------------------------------- 50 ul of 1 M MgCl2
Distilled H2O -------------------------------------- 150 ul


Second-Strand dNTP Mixture (1 ml)
10 mM dATP ------------------------------------ 100 ul of 100 mM dATP
10 mM dGTP ------------------------------------ 100 ul of 100 mM dGTP
10 mM dTTP ------------------------------------ 100 ul of 100 mM dTTP
26 mM dCTP ------------------------------------ 260 ul of 100 mM dCTP
Distilled H2O ------------------------------------- 440 ul


Blunting dNTP Mixture (1 ml)
2.5 mM dATP ----------------------------------- 25 ul of 100 mM dATP
2.5 mM dGTP ------------------------------------ 25 ul of 100 mM dGTP
2.5 mM dTTP ------------------------------------ 25 ul of 100 mM dTTP
2.5 mM dCTP ------------------------------------ 25 ul of 100 mM dCTP
Distilled H2O ------------------------------------- 900 ul


Adapters (0.4 ug/ul)
Example; EcoRI adapter, etc.


10 x Ligation Buffer (1 ml)
500 mM Tris-HCl (pH 7.4) ------------------------ 500 ul of 1 M Tris-HCl
100 mM MgCl2 ------------------------------------ 100 ul of 1 M MgCl2
10 mM Dithiothreitol (DTT) ------------------------ 10 ul of 1 M DTT
10 mM rATP -------------------------------------- 100 ul of 100 mM rATP
Distilled H2O -------------------------------------- 290 ul


XhoI Reaction Buffer (1 ml)
200 mM NaCl ------------------------------------- 40 ul of 5 M NaCl
15 mM MgCl2 ------------------------------------- 15 ul of 1 M MgCl2
Distilled H2O --------------------------------------- 945 ul


10 x STE Buffer (100 ml)
0.1 M NaCl --------------------------------------- 2 ml of 5 M NaCl
10 mM Tris-HCl (pH 8.0) ------------------------- 10 ml of 1 M Tris-HCl
1 mM EDTA (pH 8.0) ----------------------------- 0.2 ml of 0.5 M EDTA
Distilled H2O --------------------------------------- 88 ml


Lamda Vector (Lambda ZAP, ZAP Express, or SeqZAP)
Double-digest and dephosphorylate.
Vectors are digested with XhoI and a second restriction enzyme that leaves ends compatible with the adapters.


NZY Medium (1 liter)
NaCl --------------------------------------------- 5 g
MgSO4.7H20 ------------------------------------ 2 g
Yeast extract ------------------------------------- 5 g
NZ amine (casein hydrolysate) ------------------- 10 g
Agar --------------------------------------------- 15 g
(0.7% agarose for top agarose -------------------- 7 g)
Distilled H2O to make a fianl volume of --------- 1 L
Adjust the pH to 7.5 with NaOH.
Autoclave.


E. coli Host Strains (XL I -Blue MRF'or DH5a MCR)
Freshly streaked on an LB agar plate containing the appropriate antibiotic.


Packaging Extract
Gigapack Lamda packaging extract


IPTG (Isopropyl-B-D-thio-galactopyranoside)
0.5M in sterile H2O


X-Gal (5-bromo-4-chloro-3-indoyl-o-D-galactopyranoside)
250 mg/ml in Dimethyl formamide (DMF)




PROCEDURES

First-Strand cDNA Synthesis
  • First-strand cDNA synthesis should be done in RNase-free environment.

    1. In an RNase-free microfuge tube, add the reagents in the following order.
  • The final volume of the first-strand synthesis reaction should be 50 ul.

    10 x First-Strand Buffer ---------------------- 5.0 ul
    Methyl-Nucleotide Mixture ------------------ 3.0 ul
    Oligo (dT) Linker-Primer (1.5 ug/ ul) --------- 2.0 ul
    DEPC-treated H2O -------------------------- X ul
    Ribonuclease Inhibitor ----------------------- 40 U


    2. Add 5 ug of polyA+ mRNA, and mix well.

    3. Anneal Oligo (dT) Linker-Primer to the mRNA template for 10 min at room temperature.

    4. Add 250 U of Superscript II Reverse Transcriptase. The final volume of the reaction should now be 50 ul.

    5. Gently mix the sample, and briefly spin down the contents in a microcentrifuge.

    6. Incubate at 37oC for 1 hour.

    7. Place tube on ice.



    Second-Strand cDNA Synthesis

    8. To the first strand reaction (50 ul), add the following components in the following order.
  • The final volume of the second-strand synthesis reaction should be 200 ul.

    10 x Second-Strand Buffer ----------------------- 20 ul
    Second-Strand dNTP mixture -------------------- 6.0 ul
    Distilled H2O ------------------------------------- X ul
    E. coli RNase H --------------------------------- 4 U
    E. coli DNA polymerase I ----------------------- 100 U

    Briefly vortex and spin down the reaction in a microcentrifuge.

    9. Incubate for 2.5 hour at 16oC.

    10. Place tube on ice.



    Blunting the cDNA Termini

    11. Add the following reagents to the synthesized cDNA.

    Blunting dNTP Mixture --------------------------- 23 ul
    Cloned Pfu DNA polymerase --------------------- 5 U/ 2.0 ul

  • Mix well, and incubate at 70oC for 30 min. Do not exceed 30 min.

    12. Do phenol: chloroform: IAA extraction.
    Do chloroform: IAA extraction.

    13. Precipitate the cDNA by adding the following.

    3M Sodium acetate --------------------------- 20 ul
    100% Ethanol -------------------------------- 400 ul

    14. Precipitate cDNA on ice for 10 min.

    15. Centrifuge the precipitated cDNA at maximum speed and at 4oC for 1 hour.

    16. A large white pellet will form at the bottom of the microfuge tube.

    17. Wash the pellet by gently adding 500 ul of 70% ethanol, and microcentrifuge for 2 min.

    18. Aspirate the ethanol wash, and air dry the pellet.



    Ligation of the Adapters

    19. Resuspend the cDNA pellet in 9.0 ul of Adapters (0.4 ug/ ul) by gentle pipeting.

    20. Remove 1.0 ul for analysis of cDNA synthesis on a 5% nondenaturing acrylamide gel.
  • This aliquot may be frozen at -20oC.

    21. Add the following components to the tube containing the 8.0 ul of blunted cDNA and Adapters.

    10 x Ligation Buffer ------------------- 1 ul
    10 mM rATP ------------------------- 1 ul
    T4 DNA ligase ------------------------ 4 U/ ul

  • Mix well and briefly spin in a microcentrifuge.

    22. Incubate overnight at 8oC or for 2 days at 4oC.



    Phosphorylation of the Adaptors

    23. After ligation, heat-inactivate the DNA ligase by incubating at 70oC for 30 min.

    24. Spin down, and allow the reaction to cool at room temperature for 5 min.

    25. Add the following components.
  • The final volume of the phosphorylation reaction should be 25 ul.

    10 x Ligation Buffer -------------------- 1.5 ul
    10 mM rATP --------------------------- 2.0 ul
    Distilled H2O --------------------------- X ul
    T4 Polynucleotide kinase ---------------- 7 U


    26. Incubate at 37oC for 30 min.

    27. Heat-inactivate the polynucleotide kinase by incubating at 70oC for 30 min.

    28. Spin down, and allow the reaction to cool at room temperature for 5 min.



    XhoI Digestion

    29. Add the following components to the phosphorylation reaction (25 ul).
  • The final volume of the digestion reaction should be 60 ul.

    XhoI Reaction Buffer ----------------- 30 ul
    Distilled H2O ------------------------- X ul
    XhoI --------------------------------- 120 U


    30. Incubate for 1.5 hour at 37oC.

    31. Cool the reaction to room temperature.

    32. Add 15 ul of 10 x STE buffer and 75 ul distilled H2O.



    Size Fractionation

  • There are many types of filtration media used to separate DNA molecules. Sephacryl S-500 resin separates efficiently in the 2 kb size range. Drip columns made with Sephacryl S-500 resin separate by size, the larger cDNA molecules eluting from the column first and the small unligated adapters and unincorporated nucleotides eluting later.

    33. Prepare drip-column as follow.
  • Discard the plunger from a 1 ml plastic syringe, and insert a small cotton plug. Push the cotton to the bottom of the syringe.

    34. Fill the syringe to the top with Sephacryl S-500 filtration medium.

    35. Place the syringe in a rack and allow the column to drip dry.

    36. Fill the syringe up to 0.5 cm from the top with resin, and drip through.

    37. Rinse the column with four aliquots of 300 ul of 1 x STE buffer (total wash volume of 1200 ul). Drip dry after each addition of buffer.

    38. Pipet the cDNA into the washed Sephacryl S-500 drip column, and allow to drip through. This is fraction #1. The recovery volume is 150 ul and does not contain cDNA.

    39. Load two more aliquots of 150 ul of 1 x STE buffer on the column and drip through. These are fractions #2 and #3.

    40. Collect fraction #4 in a fresh tube. Load 150 ul of 1 x STE buffer and drip as before.

    41. Collect fraction #5 as in step 40. Two fractions are usually adequate (i.e. fraction #4 and #5 contain cDNAs). The size of the cDNA decreases in each additional fraction.

    42. Remove 5 ul from each fraction (or up to 1/10 of the fraction volume) for analysis of cDNA size on a 5% nondenaturing acrylamide gel. These aliquots can be frozen at -20oC.

    43. To remove any residual enzyme from previous reactions, phenol: chloroform/chloroform extract.

    44. Add twice the volume of 100% Ethanol to precipitate the cDNA.

    45. Place on ice for 1 hour.

    46. Centrifuge the fractionated cDNA at maximum speed for 30-60 min at 4oC.

    47. Wash the cDNA pellet with 200 ul of 70% Ethanol, and centrifuge for 2 min.

    48. Carefully remove the ethanol wash, and vacuum evaporate until the cDNA pellet is dry.

    49. Resuspend the cDNA in 5-10 ul of sterile water.

    50. Quantitate the cDNA by UV visualization of samples spotted on ethidium bromide agarose plates. The cDNA can be stored at -20oC.



    Ligation of the cDNA to Prepared Vector

    51. The cloning vector should be double-digested with XhoI and an enzyme which leaves ends compatible with the adapters. The vector should also be dephosphorylated to prevent vector-to-vector ligations. The final ligation reaction volume is 5 ul.

    52. To a 0.5 ml microfuge tube, add the following in order.

    Distilled H2O ------------------------------------- X ul
    10 x Ligation Buffer ------------------------------ 0.5 ul
    10 mM rATP ------------------------------------ 0.5 ul
    Double-digested Lamda Arms ------------------- 1 ug
    cDNA ------------------------------------------- 100 ng
    T4 DNA ligase ----------------------------------- 2 Weiss U/0.5 ul

    53. Incubate overnight at 4oC.



    Packaging and Plating

    54. The ligation is packaged and transfected into an appropriate E. coli host strain.

    55. Inoculate 50 ml of NZY medium with a single colony of the appropriate E. coli host. Do not add antibiotic.

    56. Grow at 30oC with gentle shaking overnight.

    57. Centrifuge the culture at 1,000g for 10 min.

    58. Gently resuspend the cells in 20 ml sterile 10 mM MgSO4.

    59. Determine the concentration of the cells by reading OD600 on a spectrophotometer.
  • Store this cell stock at 4oC for no more than 1 week.
  • To use, dilute cells to OD600 = 1.0 in 10 mM MgSO4.

    60. Package the ligation reaction following manufacturer's instructions.
  • Stop the reaction by adding 500 ul SM buffer and 20 ul chloroform.

    61. Mix the following components in a Falcon 2059 polypropylene tube.

    Diluted host cells
    200 ul
    Final packaged reaction
    1 ul


    62. Incubate the phage and the bacteria at 37oC for 15 min to allow the phage to attach to the cells.

    63. Add 2-3 mL of NZY top agarose (48oC) containing IPTG and X-gal.
  • Plate onto NZY agar plates, and place the plates upside down in a 37oC incubator.

    64. Plaques should be visible after 6-8 hours.
  • Background plaques are blue. Recombinant plaques are clear and should be 10- to 100-fold above the background.

    65. Count the plaques and calculate the titer.
  • Primary libraries can be unstable. Immediate amplification of at least a portion of the library is recommended to produce a large, stable quantity of a high-titer stock of the library.



    Amplification of the Primary cDNA Library
  • After amplification, the library is suitable for screening by a variety of techniques. More than one round of amplification is not recommended, since slower growing clones may be significantly underrepresented.

    66. Prepare the host strains.

    67. Mix aliquots of the packaged library containing 50,000 pfu (<300 ul vol) with 600 ul of host cells in Falcon 2059 polypropylene tubes.

    68. Incubate the tubes containing the phage and host cells for 15 min at 37oC.

    69. Mix 8.0 ml of melted NZY top agarose (48oC) with each aliquot of infected bacteria, and spread evenly onto a freshly poured 150 mm NZY plate.

    70. Incubate the plates at 37oC for 6-8 hours.
  • Do not allow the plaques to grow larger than 1-2 mm.

    71. Overlay the plates with 8-10 ml of SM buffer.
  • Store the plates at 4oC overnight with gentle rocking. The phage will diffuse into the SM buffer.

    72. Recover the SM buffer containing the bacteriophage from each plate, and pool it in a sterile polypropylene container.
  • Add chloroform to a 5% final concentration and mix well.

    73. Incubate for 15 min at room temperature.

    74. Remove the cell debris by centrifugation for 10 min at 500g.

    75. Recover the supernatant, and transfer it to a sterile polypropylene container.
  • Add chloroform to a 0.3% final concentration, and store at 4oC.

    76. Check the titer of the amplified library by making serial dilutions in SM buffer and plating on host cells. The average titer is usually 109-1012 PFU/ml.

    77. Frozen stocks can be made by adding DMSO to a final concentration of 7%, mixing well, and freezing at -80oC.




    NOTES

  • The mRNA must be highly purified for efficient cDNA synthesis. The mRNA solution may contain inhibitors that can be removed by phenol-chloroform extractions. The presence of DNA or rRNA will give an inaccurate concentration of mRNA leading to an insufficient amount of sample used. Treat the mRNA with RNase-free DNase, or use more mRNA sample.

  • Gel analysis may show hairpinning of the cDNA, which is caused by a number of factors: an insufficient amount of mRNA was used in the first-strand reaction, the mRNA population had tight secondary structure, the second-strand incubation temperature was higher than 16oC, or an excessive amount of DNA polymerase was used in the second-strand reaction.

  • Sephacryl S-500 drip columns can be run dry. A reservoir at the top of the column is not required. Each 150 ul wash yields 150 ul fraction volume. Fractions #1-3 can be collected in one tube since these fractions do not contain cDNA. The cDNA elutes in fractions #4 (containing fragments >1.5 kb) and #5 (containing fragments >500 bp).

  • Since the cDNA is heavily methylated, introduction into a host with an McrA, McrCB, hsdSMR, Mrr phenotype would be subject to digestion by these restriction systems. Therefore, the choice of packaging extract and an E. coli host strain is crucial.

  • Since Lamda phage can adhere to dead as well as to viable cells, the lower temperature prevents the bacteria from overgrowing.

  • Most cDNA vectors have color selection by IPTG and X-gal. These components can be added to the top agarose before plating to produce the background blue color. Use 15 ul of 0.5 M IPTG (in water) and 50 ul of X-Gal at 250 mg/ml in dimethylformamide (DMF).




    KIT INFORMATION




    REFERENCES

  • Bullock W, Fernandez JM, Short JM (1987) XL1-blue: a high efficiency plasmid transforming recA Escherichia coli strain with beta-galactosidase selection. Biotechniques 5(4): 376-379.

  • Han JH, Rutter WJ (1987) Lambda gt22, an improved lambda vector for the directional cloning of full-length cDNA. Nucleic Acids Res. 15: 6304.

  • Huynh TV, Young RA, Davis RW (1985) DNA Cloning, vol. I (Glover DA, ed.), IRL, Washington DC, pp49-78.

  • Krug MS, Berger SL (1989) First strand cDNA synthesis primed with oligo (dT). Methods Enzymol. 152: 316-325.

  • Meissner PS, Sisk W, Berman ML (1987) Bacteriophage lambda cloning system for the construction of directional cDNA libraries. PNAS USA 84: 438-447.

  • Murphy AJM, Efstratiadis A (1987) Cloning vectors for expression of cDNA libraries in mammalian cells. PNAS USA 84: 8277-8281.

  • Short JM, Fernandez JM, Sorge JA, Huse WD (1988) Lambda ZAP: a bacteriophage lambda expression vector with in vivo excision properties. Nucleic Acids Res. 16: 7583-7600.



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