Molecular Techniques and Methods

Construction of Genomic Library

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

Once high molecular weight genomic DNA has been isolated, it is necessary to reduce the size range by restriction enzyme digestion to ensure a successful genomic library construction. Restriction digestion conditions should be optimized on a small scale before performing large-scale digestion of genomic DNA for cloning. Partially digested DNA to be cloned into Xho I (or BamH I) arms of EMBL3 or LambdaGEM-11 or -12 vectors should be further size-fractionated to remove fragments less than 14 kb. This size fractionation minimizes the chance of cloning two or more DNA fragments into the same site since the maximum insert size is approximately 23 kb. Size fractionation may be accomplished by sucrose gradient centrifugation, NaCl density gradient centrifugation, or by preparative agarose gel electrophoresis followed by electroelution from the gel. Fragments to be cloned into the LambdaGEM-11 or LarnbdaGEM-12 vectors by the Xho I half-site strategy require no further size fractionation.




MATERIALS AND SOLUTIONS

Dilution Buffer (1.5 ml)
Sau3A I 10 x Buffer -------------------------- 150 ul
Acetylated BSA ------------------------------ 15 ul of Acetylated BSA (10 mg/ml)
H2O ------------------------------------------ 1335 ul


Sau3A I 10 x Buffer (1 ml)
l00 mM Tris-HCl (pH 7.5) -------------------- 100 ul of 1 M Tris-HCl
1 M NaCl ------------------------------------- 200 ul of 5 M NaCl
70 mM MgCl2 -------------------------------- 70 ul of 1 M MgCl2


Genomic DNA Assay Buffer (450 ul)
Genomic DNA (1 ug/ ul) ---------------------- 10 ul
Sau3A I 10 x Buffer --------------------------- 50 ul
Acetylated BSA (1 mg/ml) --------------------- 5 ul of Acetylated BSA (10 mg/ml)
H2O ------------------------------------------- 385 ul


Gel Loading Buffer (10 ml)
38% Sucrose ----------------------------------- 3.8 g
0.1% Bromophenol blue ------------------------ 0.2 ml of 5% BPB
67mM EDTA ---------------------------------- 1.34 ml of 0.5 M EDTA
Add distilled H2O to make a final volume of --- 10 ml


A/G Fill-in 10 x Buffer (1 ml)
0.5M Tris-HCl (pH 7.2) ------------------------ 500 ul of 1 M Tris-HCl
0.1 M MgSO4 ---------------------------------- 100 ul of 1 M MgSO4
1 mM DTT ------------------------------------- 1 ul of 1 M DTT
Acetylated BSA -------------------------------- 50 ul of BSA (10 mg/ml)
l0 mM dATP ----------------------------------- 100 ul of 100 mM dATP
l0 mM dGTP ----------------------------------- 100 ul of 100 mM dGTP
Distilled H2O ----------------------------------- 149 ul


T4 DNA Ligase 10 x Buffer (1 ml)
300 mM Tris-HCl (pH 7.8) --------------------- 300 ul of 1 M Tris-HCl
100 mM MgCl2 --------------------------------- 100 ul of 1 M MgCl2
100 mM DTT ----------------------------------- 100 ul of 1 M DTT
l0 mM ATP ------------------------------------- 100 ul of 100 mM dATP
  • Store at -20oC.


    TB Top Agar (100 ml)
    Bacto-tryptone --------------------------------- 10 g
    NaCl ------------------------------------------- 0.5 g
    Bacto-agar ------------------------------------- 0.8 g
  • Autoclave.
  • When the solution has cooled to 60oC, add 1 ml of sterile 1 M MgSO4.


    E.coli LE392
    F-, hsdR514 (rk-, mk-), supE44, supF58, lacYl or delta(lacIZY), galK2, galT22, metB l, trpR55, lamda-.
  • The bacterial strain LE392 is a permissive host strain that can be utilized for most cloning experiments.


    E.coli KW251
    F-, supE44, supF58, galK2, galT22, metB l, hsdR2, mcrB l, mcrA-, argA81:Tn10, recD 1014.
  • The bacterial strain KW251 is a permissive host strain. It has been reported that certain eukaryotic recombinants are unable to grow on or can be biased by rec+ host strains (i.e., NM538, NM539, LE392). If low cloning efficiencies are noted using conventional host strains, KW251 may be used as an alternative host. E.coli KW251 should be maintained in the presence of tetracycline.




    PROCEDURES

    Partial Digestion and Size Fractionation of Genomic DNA

    1. In order to establish the optimum enzyme concentration to generate a certain size range of DNA fragments (15-23 kb), perform the following small-scale Sau3A I enzyme reactions.

  • Prepare the following Sau3A I Dilutions on ice.
    * Sau3A I Dilutions = 10 ul of Sau3A I Preparation + X ul of Dilution Buffer

    Tube No.
    10 ul of Sau3A I
    Dilution Buffer
    Dilution Factor
    Final Enzyme Conc. (U/ ug)
    1
    Undiluted
    140 ul
    1/15
    1 U/ ug
    2
    1/15 Diluted
    90 ul
    1/150
    0.1 U/ ug
    3
    1/150 Diluted
    10 ul
    1/300
    0.05 U/ ug
    4
    1/150 Diluted
    30 ul
    1/600
    0.025 U/ ug
    5
    1/150 Diluted
    50 ul
    1/900
    0.015 U/ ug
    6
    1/150 Diluted
    70 ul
    1/1200
    0.0125 U/ ug
    7
    1/150 Diluted
    90 ul
    1/1500
    0.01 U/ ug
    8
    1/150 Diluted
    110 ul
    1/1800
    0.0085 U/ ug
    9
    1/150 Diluted
    190 ul
    1/3000
    0.005 U/ ug
    10
    1/150 Diluted
    290 ul
    1/4500
    0.0035 U/ ug


    2. Assemble small-scale digestion reactions in 10 separate tubes.
  • Add the following components in each tube.

    Genomic DNA Assay Buffer ------------------------- 45 ul
    Appropriate
    Sau3A I Dilution prepared in Step 1 ------ 5 ul


    3. Incubate at 37oC for 30 minutes.

    4. Stop the reactions by adding l ul of 0.5 M EDTA and 10 ul of Gel Loading Buffer.

    5. Load 20 ul of each reaction on a 0.4% Agarose gel along with DNA markers (Lambda DNA digested with Hind III).

    6. Run the gel at 2 volts/cm for 16-20 hours or until the bromophenol blue has just migrated off the gel.

    7. Photograph the gel and determine the amount of enzyme needed to produce the maximum intensity of fluorescence in the desired size range (15-23 kb). The intensity of fluorescence is related to the mass distribution of the DNA.
  • To obtain the maximum number of molecules in this size range for library construction, use half of the amount of enzyme that produces the maximum amount of fluorescence.


    Large-Scale Preparation of Partially Digested Genomic DNA

    8. Using the optimized conditions determined in Step 7, carry out a large-scale Sau3A I enzyme reaction with 50-100 ug of high molecular weight genomic DNA. The DNA concentration, time, and temperature should be identical to those used in the small-scale reactions.
  • Use half the number of units of Sau3A I/ ug DNA to optimize sequence representation of molecules in the desired size range.

    9. Determine the size distribution of the digestion products by removing a small aliquot of the DNA (0.5 ug) and analyzing by electrophoresis through a 0.4% Agarose gel.

    10. If the digestion is adequate, extract with 1 volume of TE-saturated phenol/chloroform.
  • Mix by gently inverting for several times and centrifuge at 12,000g for 5 minutes.

    11. Transfer the upper, aqueous phase to a fresh tube and repeat Step 10.

    12. Transfer the upper, aqueous phase to a fresh tube and add 1 volume of chloroform: isoamyl alcohol (24:1). Mix and centrifuge as in Step 10.

    13. Transfer the upper, aqueous phase to a fresh tube and add 0.5 volume of 7.5M Ammonium acetate followed by 2 volumes of 100% Ethanol. Mix and leave at -20oC for 30 minutes.

    14. Centrifuge at 12,000g for 10 minutes.

    15. Remove the supernatant, and rinse the pellet with 70% Ethanol.
  • Drain the tube and air-dry the pellet briefly.

    16. Resuspend the pellet in 500 ul of TE buffer. Store the DNA at -20oC.

    17. Partially fill-in (A/G) the Sau3A I site in genomic DNA for cloning into Xho I half-site lamda arms as follow.

    Partially digested genomic DNA -------------------- 10 ug
    A/G Fill-in 10 x Buffer ------------------------------ 5 ul
    Klenow fragment ------------------------------------ l ul/ ug DNA
    Add distilled H2O to make a final volume of ------- 50 ul

  • Incubate at 37oC for 30 minutes.


    18. Extract twice with 1 volume of TE-saturated phenol: chloroform.
  • Vortex for 1 minute and centrifuge at 12,000g for 5 minutes.

    19. Transfer the upper, aqueous phase to a fresh tube and add 1 volume of chloroform: isoamyl alcohol (24:1).
  • Vortex for 1 minute and centrifuge as in Step 18. Repeat this step.

    20. Transfer the upper, aqueous phase to a fresh tube. Add 0.5 volume of 7.5M Ammonium acetate. Add 2 volumes of 100% Ethanol and leave at -70oC for 30 minutes.
  • Centrifuge at 12,000g for 15 minutes.

    21. Carefully pour off the supernatant, wash the pellet with l ml 70% ethanol, air-dry briefly, and resuspend in 20 ul H2O (approximately 0.5 ug/ ul).
  • Determine the exact DNA concentration by absorption spectroscopy at 260 nm.
  • (1 OD260nm = 50 ng DNA/ ul)


    Ligation of Insert DNA into Lamda Vector Arms

    22. The day before the experiment, preferably late in the day, start an overnight culture of LE392 or KW251 by inoculating a single colony into 50ml of LB medium supplemented with 0.5 ml of 20% Maltose and 0.5 ml of 1 M MgSO4.
  • Shake overnight at 37oC and early the next day store at 4oC.

  • Alternatively, inoculate 50 ml of LB medium (supplemented with 0.5 ml of 20% Maltose and 0.5 ml of 1 M MgSO4) with 1 ml of an overnight culture of LE392 or KW251 cells, shake at 37oC, and store at 4oC once the OD600 nm has reached 0.6.


    23. Prepare the ligation reaction as follow.

    Lambda vector Xho I half-site arm ------------------------ 0.5 ug
    Sau 3A I partially digested genomic DNA ----------------- 0.5 ug
    T4 DNA Ligase 10 x Buffer ------------------------------- 0.5 ul
    T4 DNA Ligase (1 Weiss unit) ---------------------------- 0.5 ul
    Add distilled H2O to make a final volume of ------------- 5 ul

  • To test the background due to the vector arms alone, prepare the above reaction, but omit the insert genomic DNA.

    24. Incubate the ligation mixture(s) overnight at 4oC.


    Packaging of Ligated DNA using in vitro Packaging System

    25. Thaw the phage packaging extract on ice.

    26. Add the 1 ul of ligation reaction from step 23 to the phage packaging extract, and mix by gently tapping the bottom of the tube several times.

    27. Incubate at 22oC for 2 hours for packaging.

    28. To the packaging mix (55 ul), add SM buffer to 500 ul and 25 ul of chloroform. Mix gently by inversion and allow the chloroform to settle to the bottom of the tube.
  • The packaged phage can then be stored at 4oC for up to 3 weeks, although the titer may drop several fold under these conditions.


    Titration of Packaged Phage on LB Plates

    29. Make appropriate dilutions of the packaging extracts in SM buffer. As a general guideline, an appropriate dilution for recombinant phage is 1/1,000 or 1/10,000.
  • Add 100 ul of the diluted phage to 100 ul of prepared LE392 or KW251 bacteria and allow the phage to adsorb for 30 minutes at 37oC.

    30. Add 3 ml molten (45oC) TB Top Agar. Mix gently and immediately pour onto LB plates.

    31. Allow the top agar to harden and incubate inverted at 37oC overnight.
  • Best results are obtained by using fresh plates which have been allowed to dry overnight at room temperature so that they lose excess moisture.

    32. Count the number of plaques and calculate the titer of the phage.

    33. Continue to library screening




    NOTES

  • Fresh (or freshly thawed) T4 DNA ligase 10 x Buffer is recommended. The activity of the T4 DNA ligase can be evaluated by ligation and noting a shift in mobility (up) on a low percentage agarose gel.

  • If low titers are observed, the following alternatives are recommended.
    (a) If utilizing LE392 as the primary host strain, KW251 may be used as an alternative.
    (b) A mixing experiment may be performed in which a small aliquot of genomic insert (pre-filled reaction DNA) is mixed either with the positive control insert or lambda DNA markers. This mixture is then ligated under normal experimental conditions. A control tube containing no genomic insert and only the secondary DNA is also ligated. The samples are then loaded on a low percentage agarose gel. Inhibition of ligation efficiencies can then be noted by comparison of the two distinct samples. If ligation inhibition is observed, the genomic DNA sample should be re-extracted with phenol: chloroform, extracted with chloroform alone, and then precipitated with ethanol.
    (c) Polysaccharide contamination (observed usually in plant or bacterial genomic DNA samples) will inhibit the Klenow fill-in or the T4 DNA ligation reaction. The recommended procedure for polysaccharide removal is chloroform extraction in the presence of 1% CTAB (cetyltrimethylammonium bromide).
    (d) The genomic DNA inserts (pre-fill) may be ligated to themselves and observed on a low percentage agarose gel. If no apparent shift in mobility is noted, it is possible that the genomic DNA sample has been contaminated with an exonuclease, therefore not allowing an efficient fill-in reaction or ligation to the provided lambda arms.




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    REFERENCES



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