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.
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
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.
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.
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.