Restriction Digest and Ligation to produce Large Random Pools

Jonathan Urbach

April 6, 1998


The practical size limit of a single oligonucleotide synthesis is about 140 bases. It is however useful to create random pools that are longer than that. Generally, this involves several synthesis reactions followed by gel purification of the synthesized oligomers, PCR amplification of the pool, restriction digest of the amplified DNA, and religation to make a longer pool. Pools of length greater than 300 bases are routinely constructed this way.

Since Large Scale PCR is covered elsewhere in the compendium of Szostak Laboratory techniques, it will not be covered here. Large scale PCR reactions are generally concluded by extracting with phenol, extracting 3x with chloroform then precipitating twice with ethanol. Precipitating twice with ethanol is advisable to remove trace amounts of phenol which can inhibit restriction enzymes. DNA from large scale PCR reactions may be used without further purification in large scale restriction digests. In fact this is advisable because of the losses incurred in native gel purification.

Restriction Digests

Single and double restriction digests are often required for pool construction. Both are fairly straight-forward scaled up versions of standard molecular biology restriction digests. The main differences are that whereas in micro-scale molecular biology, restriction enzyme is often in excess of what is needed, in restriction digests on large pools, the enzyme is the limiting factor in the reaction, due to the high cost of sufficient amounts of enzyme. Generally, large scale restriction digest incubations are run overnight or up to several days.

Three Part Pool Construction

The examples used in this procedure are from the construction of the three piece CDC Pool. The individual pieces of CDC, Pool C and Pool D both contain Ban I (GGCACC) and Sty I (CCAAGG) restriction sites. Ban I and Sty I have asymmetric cut sites and are therefore directional. Although the sites can be religated, two pool fragments with identical asymmetric overhangs cannot be ligated together. This allows a much greater control over pool synthesis than one could get with a palindromic restriction site. The CDC Pool construction is outlined in figure 1.

Single Restriction Digests:

C-Sty I:

4 mL Pool C ds DNA (50 nmols, 3x1016 molecules) in 50 mM NaCl
500 無 10x New England Biolabs Buffer #3 (1)
50 無 10 mg/mL BSA
450 無 Sty I (10,000 units/ml)
5 mL Total volume

C-Ban I:

4 mL Pool C ds DNA (50 nmols, 3x1016 molecules) in 50 mM NaCl
500 無 10x Low Salt Buffer(2)
450 無 Ban I (20,000 units/ml)
4950 無 Total volume

The reactions are incubated overnight at 37°C, at which point they are essentially done (see figure 2). The progress of the reactions is monitored by electrophoresis with analytical 4% NuSeive TBE gels. Regular agarose gels with TAE do not have adequate resolution to unambiguously detect cleavage of a 14 bp fragment from small PCR DNA molecules. The reaction is concluded by adding 400 無 0.5 M Na2-EDTA, 600 無 3M NaCl, then precipitated with 2 volumes ethanol. Dried pellets are purified on 5% acrylamide native gels.

Double Digests

D-Ban I/Sty I

17 mL Pool D ds DNA (29 nmols, 1.7x1016 molecules) in 50 mM NaCl
2 mL 10x Low Salt Buffer
500 無 Ban I (20,000 units/ml)
19.5 mL Total

The reaction is incubated overnight at 37°C at which point the Ban I sites are almost completely cut, as determined by a 4% NuSeive TBE gel. The following are added to the reaction:

2.2 mL 10x New England Biolabs Buffer #3
220 無 10 mg/mL BSA
0.5 mL Sty I (10,000 units/mL)
22.42 mL Total

After incubation for 24 hours, a 4% NuSeive TBE gel ( figure 3) shows complete conversion of the starting material to the double cut Pool D fragment. The reaction is extracted with 10 mL TE-equilibrated phenol. (Further extraction with chloroform is not necessary since the residual phenol will be removed in acrylamide gel purification.) 1.0 mL 0.5 M Na2-EDTA, is added to the reaction mixture along with 2 mL 3M NaCl. The mixture was precipitated with 2 volumes ethanol and the dried pellets are purified by 5% acrylamide native gel.


DNA fragments purified on a native acrylamide gel should be relatively free from the short cleaved terminal constant region oligonucleotides. This is important because these smaller fragments when present can easily be ligated on to the random region sticky ends, which would greatly reduce the yield of full length ligated pools.

Ligation Mixture:

6 mL Pool C Ban I fragment (29 nmols in 50 mM NaCl)
6 mL Pool C Sty I fragment (28 nmols in 50 mM NaCl)
6 mL Pool D Ban I/Sty I fragment (26 nmols in 50 mM NaCl)
2 mL 10x New England Biolabs Ligase Buffer
55 無 T4 DNA Ligase (400 units/無 )
20 mL Total

Reaction progress is followed on a 4% NuSeive TBE agarose gel. The reaction is over in about 15 minutes as shown in figure 4.

The reaction is concluded by extracting with 10 mL TE-equilibrated phenol, 1 mL 0.5 M Na2-EDTA, and 2 mL 3M NaCl. The DNA is precipitated with 2 volumes ethanol. Ethanol precipitation is repeated to yield 33 nmols phenol-free DNA.

(1) New England Biolabs 10 x Buffer #3 is 1 M NaCl, 500 mM Tris HCl, 100 mM MgCl2, 10 mM dithiothreitol (pH 7.9 @ 25°C).
(2) 10x Low Salt Buffer is 200 mM Tris acetate, 100 mM magnesium acetate, 10 mM dithiothreitol (pH 7.9 @25°C). This is the same as NEB Buffer #4 minus the potassium acetate. This is used because the DNA solution in 50 mM NaCl already has enough salt for Ban I, and more salt would be deleterious.