PROMEGA ENZYME RESOURCE GUIDE

Ligases
Introduction
DNA Ligases are primarily responsible for joining the gaps that form in DNA during replication
(i.e., the joining of ‘’Okazaki’’ fragments formed by discontinuous or lagging strand replica-
tion; 1), DNA repair, and recombination. The best known RNA ligase is bacteriophage T4
RNA ligase. This enzyme does not appear to have any role in nucleic acid metabolism in bac-
teriophage T4 infected E. coli, but instead appears to be required for the attachment of the
bacteriophage’s tail fibers to its base plate during bacteriophage assembly (2). However, its
activity as a ligase has been used effectively in various molecular biology applications.
Both DNA and RNA ligases catalyze the formation of a phosphodiester bond between adja-
cent nucleotides with the concomitant hydrolysis of ATP to AMP and inorganic pyrophos-
phate. Some DNA ligases (such as E. coli DNA ligase) use nicotinamide adenine dinucleotide
(NAD) instead of ATP as a cofactor and release AMP and nicotinamide mononucleotide
(NMN) as a result of phosphodiester bond formation. In general, DNA ligases will only form
this covalent linkage in a duplex molecule, for example at a nick in double-stranded (dsDNA)
or when joining cohesive- or blunt-ended dsDNAs (Figure 1, Panel A) (3). RNA ligase, on the
other hand, has a preference for single-stranded RNA or DNA (Figure 1, Panels B and C) (4).
The ligation mechanism is essentially identical for both DNA and RNA ligases, and occurs in
three stages (Figure 3). First is the formation of an enzyme-nucleotide intermediate through
transfer of an adenylyl group (AMP) from either ATP or NAD to the epsilon-amine group of a
lysine residue in the enzyme. This results in the release of pyrophosphate when ATP is the
cofactor and NMN when NAD is used. Second, the adenylyl group is transferred from the
enzyme to the 5′-phosphate of the DNA (DNA ligases) or donor polynucleotide (RNA ligases),
thereby activating it. Third, a phosphodiester bond is formed by nucleophilic attack of the 3′-
hydroxyl group of the DNA (DNA ligases) or acceptor polynucleotide (RNA ligases) on the
activated 5′-phosphate, with concomitant release of AMP.

O O O H O

A. E – (lys) – NH2 + R – O – P – O – P – O – P – O- E – (lys) – N+ – P – O – R + PPi

O- O- O- H O-
ATP

H O
References
3′ 5′ 3′ 5′
B. E – (lys) – N+ – P – O – R + + E – (lys) – NH2
1. Okazaki, R. et al. (1968) Proc. Natl. 5′ OH O 3′ 5′ OH O 3′
O O
Acad. Sci USA 59, 598. H O- P P
2. Wood, W.B. et al. (1978) J. Biol. Chem. O- O- O O O-
253, 2437. P
3. Higgins, N.P. and Cozzarelli, R. (1989) O- O–R
In: Recombinant DNA Methodology,
Wu, R., Grossman, L. and Moldave, K.
eds., Academic Press, Inc., San Diego, O
3′ 5′
3′ 5′ O
California. C. E – (lys) – NH3+ + O- – P – O – R + H+
5′ OH O 3′ 5′ 3′
4. Uhlenbeck, O.C. and Gumport, R.I. O O–P–O
O-
(1982) In: The Enzymes, Vol. XV Part B, P
O O O- O- AMP
Boyer, P.D. ed., Academic Press, New
York, 31. P
O- O–R
2547MA01/9A

5. Zimmerman, S.B. and Pheiffer, B.H.

(1983) Proc. Natl. Acad. Sci. USA 80,
5852.
6. England, T.E., Bruce, A.G. and
Figure 3. Ligation mechanism. The three step reaction schematic for ATP-dependent DNA ligases is shown.
Uhlenbeck, O.C. (1980) Meth.
Enzymol. 65, 65. Differences for NAD-dependent ligases and RNA ligases are noted in this legend. Panel A: Transfer of an adenylyl
7. Edwards, J.B., Delort, J. and Mallet, J. group from ATP (or NAD, such as in the case of E. coli DNA ligase) to the epsilon-amine group of a lysine residue in
(1991) Nucl. Acids Res. 19, 5227. the enzyme with the concomitant release of pyrophosphate (or NMN when NAD is the adenylyl donor). Panel B: The
8. Maruyama, I.N., Rakow, T.L. and adenylyl group is transferred from the enzyme to the 5′-phosphate of the DNA. In the case of RNA ligases, it is trans-
Maruyama, H.I. (1995) Nucl. Acids ferred to the donor polynucleotide. Panel C: Nucleophilic attack by the 3′-hydroxyl group on the activated 5′-phosphate
Res. 23, 3796. group of the DNA (or acceptor polynucleotide in the case of RNA ligases) forms the phosphodiester bond, with simulta-
neous release of AMP. For Panels A-C, E = enzyme, lys = lysine residue.

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 ENZYME RESOURCE GUIDE CLONING ENZYMES

Bacteriophage T4 DNA ligase is the ligase most commonly used in the construction of recom-
binant DNA molecules for molecular biology applications. It is able to ligate DNA fragments
having either complementary cohesive or blunt ends, and has an absolute requirement for
ATP as a cofactor; it cannot use NAD. E. coli DNA ligase (which, like most prokaryotic DNA
ligases uses NAD as a cofactor instead of ATP) can sometimes be used in place of T4 DNA
ligase for ligation of single-strand breaks or joining of DNA molecules with cohesive termini.
However, unlike T4 DNA ligase, the E. coli enzyme does not show blunt-ended ligation activ-
ity, except under conditions of molecular crowding with PEG 8000 (5). For this reason T4 DNA
ligase has become more widely used in DNA manipulations (Table 1). For intermolecular liga-
tions it is important that at least one of the DNA molecules possesses a 5′-phosphate at either
end of the dsDNA in order to form a phosphodiester bond. T4 DNA ligase is also used to join
adjacent single-stranded DNA (ssDNA) molecules that have been polymerized on a template
from primers annealed at separate sites, such as during site-directed mutagenesis.
The various applications that use bacteriophage T4 RNA ligase are indicated in Table 1. This
enzyme has been used for many years to label RNA molecules at their 3′-end with a radiola-
beled nucleoside 3′,5′-bisphosphate as a complementary approach to labeling the 5′-end
using T4 polynucleotide kinase (6). More recently, this enzyme has proved useful in the
cloning of full-length cDNAs by circular and 5′-rapid amplification of cDNA ends (RACE)
(7,8). The former method involves circularization of first-strand cDNA followed by inverse
PCR, whereas the latter involves ligation of an oligonucleotide linker onto the 3′-end of the
first-strand cDNA synthesis product, followed by amplification with a primer complementary to
this linker and a gene-specific primer.

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PROMEGA ENZYME RESOURCE GUIDE

Enzyme Properties
T4 DNA Ligase
Requirements: Mg2+, ATP, DTT (or other reduc-
E.C 6.5.1.1 ing agent such as β-mercaptoethanol) (1,2).
Description Cofactor Concentration: 10mM Mg2+, 10µM-
In vivo, T4 DNA Ligase (Cat.# M1801, 1mM ATP (1), 10mM DTT (1).
M1794) catalyzes the sealing of single- Optimal Substrate: In vivo, single-stranded
stranded nicks in double-stranded DNA mol- nicks in double-stranded DNA molecules (2).
ecules (1,2). It is commonly used for the join- In vitro, two double-stranded DNAs contain-
ing of two strands of DNA between the ing either blunt (“flush”) or cohesive (“sticky”)
5′-phosphate and the 3′-hydroxyl groups of ends (1,2,5). In all cases one DNA strand
adjacent nucleotides in either a cohesive- must have a 3′-OH and the other must have
ended or blunt-ended configuration (3; a 5′-phosphate.
Figure 1, Panel A). The enzyme has also
been shown to catalyze the joining of RNA Typical Working Concentration: 0.1–1.0 units
to either a DNA or RNA strand in a duplex T4 DNA Ligase per 10µl reaction for
molecule, but will not join single-stranded cohesive-end ligations or nick sealing reac-
nucleic acids (3). tions. Up to ten-fold more ligase may be
required for blunt-ended ligations (1).
Applications
• Joining blunt-ended double-stranded DNA. Optimal pH: The optimum pH range of T4
DNA Ligase has been reported to be 7.0–7.8
• Joining cohesive-ended double-stranded (2) or 7.2–7.6 (1).
DNA.
• Sealing nicks on a DNA or RNA strand Km: 5 x 10-5–5 x 10-8M for blunt-ended DNA
annealed to a DNA or RNA (4) complemen- (1,3,6), 6 x 10-7M for cohesive ends;
tary strand. 1.5 x 10-9M for nicks (2); 1.4 x 10-8M for ATP
(1).
T4 DNA Ligase is a component of the following
Promega systems: Stimulators: Polyethylene Glycol (PEG) 6000
• Erase-a-Base® System Plus Vectors or 8000 at 5% (w/v) (or other macromole-
(Cat.# E5850) cules such as Ficoll®, albumin, or glycogen)
can stimulate ligation of blunt-ended DNAs
• Erase-a-Base® System (Cat.# E5750) over 1,000-fold, as judged by shifts in elec-
• GeneEditor™ in vitro Site-Directed trophoretic mobility. Cohesive-ended DNA
Mutagenesis System(g) (Cat.# Q9280) ligation is stimulated by PEG to a far lesser
• Altered Sites® II in vitro Mutagenesis extent. T4 RNA Ligase greatly stimulates
Systems(h) (Cat.# Q6210, Q6491, Q6090, blunt-end ligations (6).
Q6501, Q6080, Q6511) Monovalent cations at low concentration
• Altered Sites® II Mammalian in vitro (~20mM) can give slight stimulation (~30%),
Mutagenesis Systems(h) (Cat.# Q5590, but at high concentrations they will inhibit lig-
Q6600) ation (1,7). Polyamines such as spermidine
• pGEM®-T Vector Systems(d,f) (Cat.# A3600, have been reported to both stimulate (1) and
A3610) inhibit (7) ligation reactions at low (<1mM)
• pGEM®-T Easy Vector Systems(d,f) concentrations. At higher concentrations
(Cat.# A1360, A1380) polyamines inhibit ligation. HMG 14 has
been shown to stimulate blunt-ended ligation
• pTARGET™ Mammalian Expression Vector by as much as 50% when present at a 50:1
System(f,h) (Cat.# A1410) molar ratio of HMG 14:DNA (1).
• PinPoint™ Xa-1 T-Vector Systems(f,i)
(Cat.# V2610, V2850) Alternative Cofactors and Substrates: Double-
stranded DNA with blunt ends, single-
• Universal RiboClone® cDNA Synthesis stranded nicks in DNA/RNA hybrids or
System(j) (Cat.# C4360) RNA/RNA hybrids. In all cases, one DNA or
RNA strand must have a 3′-OH and the other
must have a 5′-phosphate. Mn2+ can substi-
tute for Mg2+ with reduced efficiency (1).
Inhibitors: dATP is a competitive inhibitor of
ATP in ligation reactions (2). Monovalent
cations inhibit ligations with almost no activity
seen at greater than 200mM (1,7).

10 T W O
 ENZYME RESOURCE GUIDE CLONING ENZYMES

There are conflicting reports concerning Contaminant Assays

polyamines such as spermidine, which are Endonuclease Assay: 1µg of pGEM®-5Zf(+)
reported to both stimulate (1) and inhibit (7) Vector is incubated with 5 units of T4 DNA
ligation reactions at low (<1mM) concentra- Ligase in T4 DNA Ligase 1X Buffer (Tables
tions. At higher concentrations they inhibit 12,15) for 16 hours at 37°C. Following incu-
ligation (1,7). Ethidium bromide inhibits liga- bation the DNA is visualized on an ethidium
tions with an ID50 of approximately 4.3µM (1). bromide-stained agarose gel to verify the
Anions are reported to inhibit blunt-end liga- absence of visible nicking or cutting.
tions at concentrations greater than 25mM
for phosphates and greater than 50mM for Single-Stranded and Double-Stranded DNase
salts in general (1). Hexaminecobalt chloride Assay: To test for DNase activity, 50ng of
and Cibacron blue F3GA act as inhibitors radiolabeled single-stranded or double-
(1). Excess ATP has been reported to inhibit stranded DNA is incubated with 20 units of
blunt-ended ligations at high concentration T4 DNA Ligase in 1X Buffer (Tables 12,15)
(5mM) (5). for 16 hours at 37°C. Minimum passing spec-
ification is <2% release of single-stranded
Ki: 3.5 x 10-5M for dATP (2). and <1% release of double-stranded radiola-
Temperature Stability: T4 DNA Ligase is inacti- beled nucleotides as monitored by scintilla-
vated at temperatures above 37°C. tion counting of TCA-soluble material.

Inactivation: Incubate at 70°C for 10 minutes RNase Assay: To test for RNase activity, 50ng
or add EDTA to 25mM concentration. of radiolabeled RNA is incubated with 20
units of T4 DNA Ligase in 1X Buffer (Tables
Genetic Locus: Bacteriophage T4 gene 30 (1). 12,15) for 5 hours at 37°C. Minimum passing
specification is <3% release of radiolabeled
Promega Product Information nucleotides as monitored by scintillation
Source: Purified from an E. coli strain express- counting of TCA-soluble material.
ing a recombinant clone. Blue/White Cloning Assay: This assay is
Molecular Weight: 68kDa. performed to demonstrate that T4 DNA
Ligase is free from contaminating activities,
Typical Working Conditions: A typical ligation of which can affect the efficiency and integrity
cohesive-ended DNA contains 10–200ng of of plasmid cloning. Any exonuclease or
vector DNA and sufficient insert DNA to polymerase activity that alters the termini of
make a 1:1, 1:3 or 3:1 insert:vector molar linearized plasmids during ligation will result
ratio. The DNA is incubated in buffer contain- in a proportion of white-colored colonies
ing 30mM Tris-HCl (pH 7.8), 10mM MgCl2, above background levels.
10mM DTT and 1mM ATP in a final volume of
10µl. 0.1–1 unit of T4 DNA Ligase is added. A pGEM® series Vector is linearized with
For cohesive-end ligations, incubate at three different restriction enzymes in sepa-
20–25°C for approximately 3 hours or at rate reactions to generate three different
4–8°C overnight. For blunt-end ligations types of termini: EcoR I for 5′-overhangs,
incubate at 15–20°C overnight. Kpn I for 3′-overhangs, and Hinc II for blunt
ends. Linearized plasmids are purified, and References
Storage Conditions: Store at –20°C. T4 DNA ligations are performed using 12 units of T4 1. Eun, H.M. (1996) Enzymology Primer
Ligase is supplied in storage buffer contain- DNA Ligase in overnight incubations at 4°C. for Recombinant DNA Technology,
ing 10mM Tris-HCl (pH 7.4), 50mM KCl, 1mM Competent JM109 cells are transformed with Academic Press, Inc., San Diego,
DTT, 0.1mM EDTA and 50% glycerol. ligated plasmids and plated on X-Gal/IPTG/ California.
Unit Definition: 0.01 Weiss unit of T4 DNA Amp plates. A minimum of 750 colonies is
counted. White colonies result from trans- 2. Weiss, B. et al. (1968) J. Biol. Chem.
Ligase is the amount of enzyme required to 243, 4543.
catalyze the ligation of greater than 95% of formation with ligated plasmids that have
damaged ends. These white colonies repre- 3. Higgins, N.P. and Cozzarelli, R. (1989)
the Hind III fragments of 1µg of Lambda In: Recombinant DNA Methodology.
DNA at 16°C in 20 minutes. (One Weiss unit sent the number of false positives expected
in a typical cloning experiment. Enzymes that Wu, R., Grossman, L., Moldave, K.
is the amount of enzyme that catalyzes the eds., Academic Press, Inc., San Diego,
conversion of 1nmol of 32PPi into a charcoal- generate overhangs must produce fewer
than 2% white colonies and blunt-cutting California.
absorbable form in 20 minutes at 37°C in an
ATP-PPi exchange form.) enzymes must produce less than 5% white 4. Engler, M.J. and Richardson, C.C.
colonies. The minimum transformation effi- (1982) In: The Enzymes, Vol. 15, Boyer,
Purity: The purity is ≥90% as judged by SDS- ciency must be 1 x 105cfu/µg DNA. Figure 4 P.D. ed., Academic Press, New York,
polyacrylamide gels with Coomassie® blue includes a diagram of the blue/white color New York.
staining. selection protocol. 5. Sambrook, J., Fritsch, E.F. and
Activity Assays Maniatis, T. (1989) Molecular Cloning:
A Laboratory Manual, Cold Spring
Lambda Packaging Efficiency: A control insert is
Harbor Laboratory, Cold Spring Harbor,
ligated to 1µg of EMBL3 Vector Arms with 2.5
New York.
units of T4 DNA Ligase. The ligated DNA is
packaged using Packagene® Extract (Cat.# 6. Sugino, A. et al. (1977) J. Biol. Chem.
K3151). The packaged phage is diluted 252, 3987.
1:10,000 and used to infect bacterial strain 7. Raae, A.J. et al. (1975) Eur. J.
LE392. After an overnight incubation at 37°C, Biochem. 60, 437.
the phage titer and the packaging efficiency
are measured. The minimum packaging effi-
ciency must be 5 x 106pfu/µg DNA.

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 PROMEGA ENZYME RESOURCE GUIDE

Applications
T4 RNA Ligase • Labeling the 3′-end of RNA with cytidine
E.C. 6.5.1.3 3′,5′-bisphosphate (10,11).
• Cloning of full-length cDNAs/5′-RACE
Description (14,15).
T4 RNA Ligase (Cat.# M1051) catalyzes the • Intra- and intermolecular ligation of single-
ATP-dependent formation of either an intra- stranded DNA, RNA and oligonucleotides
or intermolecular 3′,5′-phosphodiester bond (2,7–9,21).
between a donor poly- or oligonucleotide
containing a 5′-phosphate group and an • Incorporation of unnatural amino acids and
acceptor poly- or oligonucleotide containing labels into proteins (16–19).
a 3′-hydroxyl group (1,2). The minimal length • Stimulation of blunt-ended ligation effi-
of a polyribonucleotide for circularization is 8 ciency (20).
bases (3). For an intermolecular reaction the
minimum size for an acceptor is a trinucleo- Enzyme Properties
tide, whereas the donor can be as small as a Requirements: Both Mg2+ and ATP are
nucleoside 3′,5′-bisphosphate (4–6). This required for enzyme activity.
enzyme is used to circularize RNA molecules
or join them to other RNAs. DNAs can also Cofactor Concentration: A concentration of
be used in intra- and intermolecular reac- 5–10mM Mg2+ is optimal for activity.
tions, although with less efficiency than RNA Concentrations above 10mM are inhibitory
(7–9). (9,22). Although a final ATP concentration of
1mM is used in the unit activity assay (circu-
T4 RNA Ligase is used to specifically label larization of 5′-[32P]-rA14–20) of T4 RNA
the 3′-end of RNA molecules with 5′-32P- Ligase, 100µM ATP is sufficient for ligase
radiolabeled nucleoside 3′,5′-bisphosphate reactions (1,9,22).
(e.g., [5′-32P]pCp) (10,11). The resulting
3′-end labeled RNAs can be used for enzy- Optimal Substrate: For intramolecular circular-
matic or chemical sequencing studies or for ization reactions, the optimal substrate is a
studies of ribonuclease activity and RNA/pro- polynucleotide with a 5′-phosphate and a
tein interactions (12,13). T4 RNA Ligase can 3′-hydroxyl group. For intermolecular reac-
also be used in 5′-RACE (Rapid Amplification tions, the donor should possess a 5′-phos-
References of cDNA Ends) to ligate a specific oligo- phate and the acceptor a 3′-hydroxyl. To limit
deoxyribonucleotide to the 3′-end of the first the reaction to a single ligation product, the
1. Silber, R., Malathi, V.G. and Hurwitz, J. acceptor should possess a hydroxyl group
strand of cDNA synthesis. The oligodeoxy-
(1972) Proc. Natl. Acad. Sci. USA 69, at both the 5′- and 3′-termini, whereas the
ribonucleotide serves as a primer binding
3009. donor should possess a 5′- and 3′-phos-
site for the upstream primer in 5′-RACE (14).
2. Uhlenbeck, O.C. and Gumport, R.I. Alternatively, the cDNA may be circularized phate. For a single nucleotide donor, the
(1982) In: The Enzymes, Vol. XV Part B, in an intramolecular reaction for use in circu- reaction can be driven towards high yields of
Boyer, P.D. ed., Academic Press, New lar RACE (cRACE) (15). product by incubating with a molar excess of
York, 31. donor over acceptor (7,10). In contrast, the
3. Kaufmann, G., Klein, T. and Littauer, T4 RNA Ligase has also been used for the use of oligonucleotide donors requires an
U.Z. (1974) FEBS Lett. 46, 271. site-specific incorporation of unnatural amino excess of acceptor to donor. A molar ratio of
acids into protein (16,17). This involves using 5:1 is usually optimal (7,8).
4. Hinton, D.M., Baez, J.A. and Gumport, T4 RNA Ligase to ligate a CA dinucleotide
R.I. (1978) Biochemistry 17, 5091. modified with an unnatural amino acid onto Typical Working Concentration: 100 units per
5. Higgins, N.P., Geballe, A.P. and the 3′-terminus of a 3′-CA deficient amber milliliter of reaction (10).
Cozzarelli, N.R. (1979) Nucl. Acids suppressor tRNA. Using this method, it is Optimal pH: pH 7.5–8.2 (Tris-HCl, 25°C) (1).
Res. 6, 1013. possible to introduce a variety of labels at
6. England, T.E. and Uhlenbeck, O.C. specific sites in a protein (18,19). Km: For ATP, 12µM (22).
(1978) Biochemistry 17, 2069. Isoelectric Point: pI= 6.1.
Another useful application of T4 RNA Ligase
7. Brennan, C.A., Manthey, A.E. and involves the blunt-end ligation of double-
Gumport, R.I. (1983) Meth. Enzymol. Stimulators: PEG 8000 increases the ligation
stranded DNA. Although T4 RNA Ligase efficiency of single-stranded DNA 30-fold at
100, 38. cannot by itself catalyze this reaction, it can a concentration of 25%, whereas the addition
8. Tessier, D.C., Brousseau, R. and stimulate the activity of T4 DNA Ligase in of dimethyl sulfoxide (DMSO) to a final con-
Vernet, T. (1986) Anal. Biochem. 158, joining blunt ends by as much as 20-fold centration of 10–20% increases the yield of
171. (20). RNA ligations 2- to 3-fold (8,11).
9. Sugino, A., Snoper, T.J. and Cozzarelli,
N.R. (1977) J. Biol. Chem. 252, 1732.
10. England, T.E., Bruce, A.G. and
Uhlenbeck, O.C. (1980) Meth.
Enzymol. 65, 65.
11. England, T.E. and Uhlenbeck, O.C.
(1978) Nature 275, 560.
12. Peattie, D.A. (1979) Proc. Natl. Acad.
Sci. USA 76, 1760.

12 T W O
 ENZYME RESOURCE GUIDE CLONING ENZYMES

Alternative Cofactors and Substrates: T4 RNA Activity Assay

Ligase can utilize a wide variety of modified Functional Assay: The RNA substrate (5′-[32P]-
nucleoside 3′,5′-bisphosphates as donors in rA14–20 , 10µM of 5′-termini) is ligated in the
ligation reactions. Examples include 5-bro- presence of T4 RNA Ligase 1X Buffer (Table
modeoxyuridine, 2′-O-methylcytidine, and 15) and T4 RNA Ligase for 15 minutes at
1-methylguanosine (2). 37°C. After ligation, the reaction is termi-
Inhibitors: The nucleoside 2′,5′-bisphosphate, nated by heating at 100°C for 2 minutes. The
2′,5′-ADP inhibits T4 RNA Ligase activity in ligated substrate is then treated with 10 units
the presence of magnesium (23). of Calf Intestinal Alkaline Phosphatase (Cat.#
M1821) for 10 minutes at 37°C. The amount
Inactivation: 65°C for 15 minutes or 100°C for of phosphatase-resistant substrate is moni-
2 minutes (1,9,22). tored by scintillation counting of the TCA-pre-
cipitable material.
Genetic Locus: Bacteriophage T4 gene 63 (24).
Contaminant Assays
Promega Product Information DNase Assay: To test for the absence of
Source: Purified from an E. coli strain DNase activity, 50ng of radiolabeled DNA is
expressing a recombinant clone. incubated with 20 units of T4 RNA Ligase in
T4 RNA Ligase 1X Buffer (Tables 12, 15) for
Molecular Weight: T4 RNA Ligase is a 3 hours at 37°C. The minimum passing spec-
monomeric enzyme with a molecular weight ification is <1% release of radiolabeled
of 43.5kDa (24). nucleotides as monitored by scintillation
Typical Working Conditions: For ligation of sin- counting of TCA-soluble material.
gle-stranded nucleic acids to each other use RNase Assay: To test for the absence of
the T4 RNA Ligase 10X Buffer supplied with RNase activity, 50ng of radiolabeled RNA is
the enzyme, diluted 1:10 (50mM Tris (pH incubated with 20 units of T4 RNA Ligase in
7.8), 10mM MgCl2, 5mM DTT and 1mM ATP) T4 RNA Ligase 1X Buffer (Tables 12, 15) for
and BSA at a final concentration of 10µg/ml. 3 hours at 37°C. The minimum passing spec-
Incubate at 17–25°C for 10–16 hours ification is <1% release of radiolabeled
(8,14,15,21). Labeling the 3′-end of RNA nucleotides as monitored by scintillation
molecules with cytidine 3′,5′-[5′-32P] bisphos- counting of TCA-soluble material.
phate can be carried out in the same buffer,
but reactions are incubated at 5°C for 6–24 Endonuclease Assay: To test for endonuclease
hours and DMSO is added to 10% final con- activity, 1µg of lambda or pGEM®(d) DNA is
centration (10,11). incubated with 20 units of T4 RNA Ligase in
T4 RNA Ligase 1X Buffer (Tables 12, 15) for
Storage Conditions: Store at –20°C. T4 RNA 3 hours at 37°C. Following incubation, the
Ligase is supplied in storage buffer contain- DNA is visualized on an ethidium bromide-
ing 10mM Tris (pH 7.5), 50mM KCl, 0.1mM stained agarose gel to verify the absence of References (continued)
EDTA, 1mM DTT, 50% glycerol and 0.1% visible nicking or cutting.
Tween® 20. 13. Caruccio, N. and Ross, J. (1994) J.
Biol. Chem. 269, 31814.
Unit Definition: One unit is defined as the
amount of enzyme required to catalyze the 14. Edwards, J.B., Delort, J. and Mallet, J.
formation of 1 nanomole of 5′-[32P]-rA14–20 (1991) Nucl. Acids Res. 19, 5227.
into a phosphatase-resistant form in 30 min- 15. Maruyama, I.N., Rakow, T.L. and
utes at 37°C at a 5′-terminal concentration of Maruyama, H.I. (1995) Nucl. Acids
10µM. The reaction conditions are specified Res. 23, 3796.
below under Functional Assay. 16. Noren, C.J. et al. (1989) Science 244,
Purity: T4 RNA Ligase is determined to be 182.
>90% homogeneous, as judged by SDS- 17. Noren, C.J. et al. (1990) Nucl. Acids
polyacrylamide gels with Coomassie® blue Res. 18, 83.
staining. 18. Cornish, V.W. et al. (1994) Proc. Natl.
Acad. Sci. USA 91, 2910.
19. Nowak, M.W. et al. (1995) Science 268,
439.
20. Sugino, A. et al. (1977) J. Biol. Chem.
252, 3987.
21. Romaniuk, P.J. and Uhlenbeck, O.C.
(1983) Meth. Enzymol. 100, 52.
22. Cranston, J.W. et al. (1974) J. Biol.
Chem. 249, 7447.
23. Sugiura, M. et al. (1979) FEBS Lett. 97,
73.
24. Rand, K.N. and Gait, M.J. (1984)
EMBO J. 3, 397.

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 PROMEGA ENZYME RESOURCE GUIDE

Blue/White Color Selection

Blue/White Color Selection Tips 1. Pre-chill sterile 17 x 100mm polypropylene
Xmn I 1994
culture tubes (e.g., Falcon 2059) on ice, one

➞
Sca I 1875 T7
1 start
Cells used for blue/white color selection have f1 ori
Apa I 14 per transformation.
Aat II 20
a deletion of the lacZ gene (∆lac in geno- Sph I
Nco I
26
37
type). This mutation results in the production Sac II 46 multiple 2. Remove frozen competent cells from –70°C
Amp r pGEM-5Zf(+/-) EcoR V 51

of nonfunctional β-galactosidase. However, if and place on ice for 5 minutes or until just
lacZ Spe I 55 cloning
Vectors Not I 62
Pst I 73 region
the deleted portion of β-galactosidase is sup- Sal I
Nde I
75
82
thawed. Competent cells will quickly lose their
plied by a lacZ-containing plasmid, β-galac- competency if warmed above 4°C.
Sac I 94
BstX I 103
Nsi I 112
tosidase activity can be restored. This 126

➞
ori SP6
3. Gently mix the cells by flicking the tube and
process is known as α-complementation. transfer 100µl of the thawed competent cells to
Cells that contain an intact lacZ gene (e.g., each of the pre-chilled culture tubes.
HB101) cannot be used for blue/white color
selection. Standard Cloning Experiment 4. Add 1–50ng of ligated DNA (in a volume not
1. Linearize plasmid and purify greater than 10µl) to competent cells.
◆ To determine the transformation efficiency Transformation efficiency will decrease with
2. Ligate with insert
of your cells, see Promega’s Protocols and greater amounts of DNA. Move the pipette tip
Applications Guide, Third Edition, page 46, Add 1-50ng of ligated DNA through the cells while dispensing. Quickly
or Technical Bulletin #TB095. or approximately 1µl of flick the tube several times. Do not pipet or
◆ LB medium can be used in place of SOC ligation reaction to 100µl vortex to mix.
freshly thawed competent
medium if desired. In our experience the
cells on ice. Mix and 5. Immediately return the tubes to ice for 10 min-
use of SOC medium results in maximum incubate on ice for 10
transformation efficiencies. utes.
minutes.
◆ Perform a mock transformation of compe- 6. Heat-shock the cells for 45–50 seconds in a
tent cells to which no DNA is added. No water bath at exactly 42°C. DO NOT SHAKE.
colonies should result. Presence of Heat-shock cells at 42˚C for
45-50 seconds. Plate on ice 7. Immediately place the tubes on ice for 2 min-
colonies may be the result of inactive for 2 minutes. Add 900µl of utes.
antibiotic or contaminated cells. SOC or LB medium and
◆ Light blue colonies? These colonies may incubate 60 minutes at 37˚C. 8. Add 900µl of cold (4°C) SOC or LB medium to
contain inserts. The causes and cures for each transformation reaction then incubate for
light blue colonies are discussed in 900µl of SOC or LB medium 60 minutes at 37°C with shaking (approxi-
Promega Notes 41. mately 225rpm).
◆ It is possible for an insert to ligate into a Plate 100µl of undiluted, 1:10 9. For each transformation reaction, we recom-
vector in-frame and not disrupt the lacZ and 1:100 dilutions of cells mend diluting the cells 1:10 and 1:100 in
gene. This is more likely to happen with on LB/IPTG/AMP plates. medium, and plating 100µl of the undiluted,
short inserts. In such a case the dephos- Incubate at 37˚C overnight. 1:10 and 1:100 dilutions on antibiotic plates.
phorylated vector-only control will contain The 1:100 dilution may be omitted for transfor-
few colonies but the insert-plus-vector mations from ligation reactions. Plate the trans-
plate will contain many blue colonies. formed cells on LB plates containing100µg/ml
◆ An alternative to preparing plates contain- ampicillin, 0.1mM IPTG and 40µg/ml X-Gal.
ing X-Gal and IPTG is to spread 20µl of Most plasmids used for routine subcloning
50mg/ml X-Gal and 100µl of 0.1M IPTG contain the gene for β-lactamase (Amp r ) and
onto previously prepared LB/antibiotic White Colony Blue Colony
(successful (vector
should be selected for using liquid and solid
plates. Allow these components to absorb medium containing 100µg/ml ampicillin. Other
recombinant) religation)
for at least 30 minutes (or until the plate common selectable markers include tetracy-
surface appears dry) at 37°C prior to plat- cline (use at 12.5µg/ml on plates, 10µg/ml in
ing cells. liquid culture), chloramphenicol (use at
◆ When cloning PCR products, the desired Insertion of a 20µg/ml) and kanamycin (use at 30µg/ml).
PCR product should be purified away from foreign DNA
GC
GC
GC
No GC
GC
GC
Antibiotics are heat sensitive; do not add to
nonspecific products, especially primer- fragment and CG
CG
CG
disruption CG
CG
CG culture medium above 55°C.
disruption of of lacZ
dimers. Primer-dimers ligate efficiently and
2617MA04/9A

lacZ 10. Incubate the plates at 37°C for 12–14 hours

will generate many white colonies that
(overnight).
appear to contain no insert.
◆ Only cells that overexpress the lac repres- 11. Colonies that contain recombinant plasmid
sor (lacI q in genotype) need to be plated (disruption of lacZ α-peptide) will appear white
Figure 4. Blue/white color selection protocol. while colonies that contain nonrecombinant
on IPTG-containing plates. IPTG inactivates
the lac repressor allowing the lac promoter plasmid (intact lacZ α-peptide) will appear
to function. Overexpression of the lac blue.
repressor is desirable because it allows
control of expression of plasmids contain-
ing lac or tac promoters. Further information on bacterial transformation and
◆ In JM109 cells, the lacZ gene harboring a blue/white color selection can be found in
deletion of the α-peptide, which is required Promega’s Protocols and Applications Guide,
for blue/white color selection, is located on Third Edition (pp. 46, 51, 383), and in Promega
the F′ episome. JM109 cells used to pre- Technical Bulletin #TB095.
pare competent cells should be maintained
on M9 minimal medium to prevent loss of
the F′ episome. White colonies that do not
contain inserts are generally the result of
loss of the F′ episome.

14 T W O