1
Minding your caps and tails –
considerations for functional
mRNA synthesis
Applications of synthetic mRNA have grown and become considerably diversified
in recent years. Examples include the generation of pluripotent stem cells (1-3),
vaccines and therapeutics (4-5), and CRISPR/Cas9 genome editing applications
(6-8). The basic requirements for a functional mRNA – a 7-methylguanylate
cap at the 5´ end and a poly(A) tail at the 3´ end – must be added in order to
obtain efficient translation in eukaryotic cells. Additional considerations can
include the incorporation of internal modified bases, modified cap structures and
polyadenylation strategies. Strategies for in vitro synthesis of mRNA vary according
to the desired scale of synthesis. This article discusses options for the selection of
reagents and the extent to which they influence synthesized mRNA functionality.
by Breton Hornblower, Ph.D., G. Brett Robb, Ph.D. and George Tzertzinis, Ph.D.,
New England Biolabs, Inc.
FEATURE ARTICLE
be INSPIRED
drive DISCOVERY
stay GENUINE
continued on page 2...
A nascent mRNA, synthesized in the nucleus,
undergoes dierent modifications before it can
be translated into proteins in the cytoplasm. For
a mRNA to be functional, it requires modified
5´ and 3´ ends and a coding region (i.e., an
open reading frame (ORF) encoding for the
protein of interest) flanked by the untranslated
regions (UTRs). The nascent mRNA (pre-mRNA)
undergoes two significant modifications in addition
to splicing. During synthesis, a 7-methylguanylate
structure, also known as a “cap”, is added to the 5´
end of the pre-mRNA, via 5´ → 5´ triphosphate
linkage. This cap protects the mature mRNA from
degradation, and also serves a role in nuclear
export and ecient translation.
The second modification occurs post-
transcriptionally at the 3´ end of the nascent
RNA molecule, and is characterized by addition
of approximately 200 adenylate nucleotides
(poly(A) tail). The addition of the the poly(A) tail
confers stability to the mRNA, aids in the export
of the mRNA to the cytosol, and is involved
in the formation of a translation-competent
ribonucleoprotein (RNP), together with the 5´
cap structure. The mature mRNA forms a circular
structure (closed-loop) by bridging the cap to the
poly(A) tail via the cap-binding protein eIF4E
(eukaryotic initiation factor 4E) and the poly(A)-
binding protein, both of which interact with eIF4G
(eukaryotic initiation factor 4G), (Figure 1, (9)).
RNA can be eciently synthesized in vitro (by
invitro transcription, IVT) with prokaryotic phage
polymerases, such as T7, T3 and SP6. The cap
and poly(A) tail structures characteristic of mature
mRNA can be added during or after the synthesis
by enzymatic reactions with capping enzymes and
Poly(A) Polymerase (NEB #M0276), respectively.
There are several factors to consider when planning
for IVT-mRNA synthesis that will influence
the ease-of-experimental setup and yield of the
final mRNA product. These are discussed in the
following sections.
DNA TEMPLATE
The DNA template provides the sequence to be
transcribed downstream of an RNA polymerase
promoter. There are two strategies for generating
transcription templates: PCR amplification and
linearization of plasmid with a restriction enzyme
(Figure 2). Which one to choose will depend on
the downstream application. In general, if multiple
sequences are to be made and transcribed in parallel,
PCR amplification is recommended as it generates
many templates quickly. On the other hand, if large
amounts of one or a few templates are required,
plasmid DNA is recommended, because of the
relative ease of producing large quantities of high
FIGURE 1:
Translation initiation complex
A mature mRNA, consisting of the 5´ and 3´ untranslated regions (UTRs)
and the open reading frame (ORF), forms a “closed-loop” structure via
interactions mediated by protein complexes that bind the cap structure
and the poly(A) tail.
40S
AAAAAAAAAAAA
3´ UTR
5´ UTR
ORF
eIF4E
Cap
Poly(A) Binding Proteins
(PABPs)
FIGURE 2:
Methods for generating transcription templates
(A) PCR can be used to amplify target DNA prior to transcription. A promoter can be introduced via the upstream primer.
(B) When using plasmid DNA as a template, linearize with an enzyme that produces blunt or 5´-overhanging ends. Using a type IIS restriction
enzyme (e.g., BspQI) allows RNA synthesis with no additional 3´-nucleotide sequence from the restriction site.
5´
3´
3´
5´
5´
3´
3´
5´
5´
3´
3´
5´
3´
5´
3´ 5´
Primer
Primer
Target DNA
PCR
5´
3´
3´
5´
A. PCR-Based Strategy B. Blunt Versus Type IIS Enzyme-Based Strategies
Blunt
digestion
Digestion
with BspQI
NNNNNGAAGAGC
NNNNNCTTCTCG
BspQI
5´...
3´...
...3´
...5´
NNNNNNNNNN
NNNNNNNNNN
5´...
3´...
...3´
...5´
Target
DNA
5´
3´
3´
5´
DNA template
5´...
3´...
3´
5´
5´...
3´...
3´
5´
RNA
polymerase
promoter
continued on page 3...
2
quality, fully characterized plasmids. There are
dierent versions of plasmids available that allow
for propagation of homopolymeric A-tails of
defined length (1).
PCR allows conversion of any DNA fragment to
a transcription template by appending the T7 (or
SP6) promoter to the forward primer (Figure 2A).
Additionally, poly(d)T-tailed reverse primers can
be used in PCR to generate transcription templates
with A-tails. This obviates the need for a separate
polyadenylation step following transcription.
Repeated amplifications should, however, be
avoided to prevent PCR-generated point mutations.
Amplification using PCR enzymes with the highest
possible fidelity, such as Q5
®
High-Fidelity DNA
Polymerase (NEB #M0491), reduces the likelihood
of introducing such mutations (2).
The quality of the PCR reaction can be assessed
by running a small amount on an agarose gel, and
DNA should be purified before in vitro transcription
using a spin column or magnetic beads (e.g.,
AMPure
®
beads). Multiple PCR reactions can be
purified and combined to generate a DNA stock
solution that can be stored at -20°C and used as
needed for in vitro transcription.
Plasmid templates are convenient if the template
sequence already exists in a eukaryotic expression
vector also containing the T7 promoter (e.g.,
pcDNA vector series). These templates include
5´- and 3´-untranslated regions (UTR), which are
important for the expression characteristics of the
mRNA.
Plasmid DNA should be purified and linearized
downstream of the desired sequence, preferably
with a restriction enzyme that leaves blunt or 5´
overhangs at the 3´ end of the template. These
are favorable for proper run-o transcription by
T7 RNA Polymerase (NEB #M0274), while 3´
overhangs may result in unwanted transcription
products. To avoid adding extra nucleotides from
the restriction site to the RNA sequence, a Type
IIS restriction enzyme can be used (e.g., BspQI,
NEB #R0712), which positions the recognition
sequence outside of the transcribed sequence
(Figure 2B, page 2). The plasmid DNA should be
completely digested with the restriction enzyme,
followed by purification using a spin column
(e.g., Monarch
®
PCR & DNA Cleanup Kit (5μg)
NEB #T1030) or phenol extraction/ethanol
precipitation. Although linearization of plasmid
involves multiple steps, the process is easier to scale
for the generation of large amounts of template for
multiple transcription reactions.
IN VITRO TRANSCRIPTION
There are two options for the in vitro transcription
(IVT) reaction depending on the capping strategy
chosen: standard synthesis with enzyme-based
capping following the transcription reaction (post-
transcriptional capping) or incorporation of a cap
analog during transcription (co-transcriptional
capping) (Figure 3). Method selection will depend
on the scale of mRNA synthesis required and
number of templates to be transcribed.
FIGURE 3:
In vitro transcription options based upon capping strategy
Enzyme-based capping (top) is performed after in vitro transcription using 5´-triphosphate RNA, GTP, and S-adenosyl- methionine (SAM).
Cap-0 mRNA can be converted to Cap-1 mRNA using mRNA cap 2´-O-methyltransferase (MTase) and SAM in a subsequent or concurrent
reaction. The methyl group transferred by the MTase to the 2´-O of the first nucleotide of the transcript is indicated in red. Conversion of ~100%
of 5´-triphosphorylated transcripts to capped mRNA is routinely achievable using enzyme-based capping.
Co-transcriptional capping (bottom) uses an mRNA cap analog, shown in yellow, in the transcription reaction. For ARCA (anti-reverse cap
analog) (left),the cap analog is incorporated as the first nucleotide of the transcript. ARCA contains an additional 3´-O-methyl group on the
7-methylguanosine to ensure incorporation in the correct orientation. The 3´-O-methyl modification does not occur in natural mRNA caps.
Compared to reactions not containing cap analog, transcription yields are lower. ARCA- capped mRNA can be converted to cap 1 mRNA using
mRNA cap 2´-O-MTase and SAM in a subsequent reaction. CleanCap Reagent AG (right) uses a trinucleotide cap analog that requires a
modified template initiation sequence. A natural Cap-1 structure is accomplished in a co-transcriptional reaction.
DNA template
RNA polymerase
promoter
5´
3´
3´
5´
P
P
P
m
7
G
CO-TRANSCRIPTIONAL mRNA CAPPING
With Anti-Reverse Cap Analog (ARCA)
+
ARCA
RNA polymerase
CleanCap
Reagent AG
mRNA Cap
2´-O-Methyltransferase
3´
Cap-0 mRNA
5´
SAM
3´
Uncapped RNA transcript
5´
P
P
P
3´
Cap-1 mRNA
5´
P
P
P
m
7
G
2´-O-methylation
3´-O-methylation
OCH
3
OCH
3
OCH
3
OCH
3
NTPs
RNA polymerase
NTPs
With CleanCap
®
Reagent AG
3´
Cap-1 mRNA
5´
P
P
P
m
7
G
OCH
3
P
P
P
m
7
G
A G
OCH
3
G
P
P
P
m
7
G
GG A G
DNA template
RNA polymerase
promoter
5´
3´
3´
5´
2´-O-methylation
POST-TRANSCRIPTIONAL ENZYME-BASED mRNA CAPPING
With Vaccinia Capping System With Vaccinia Capping System and
mRNA Cap 2´-O-Methyltransferase
Vaccinia Capping
Enzyme
RNA polymerase + NTPs
GTP
SAM
3´
RNA transcript
5´
P
P
P
3´
Cap-0 mRNA
5´
P
P
P
m
7
G
P
P
P
G
Phosphate
DNA template
RNA polymerase
promoter
5´
3´
3´
5´
mRNA Cap
2´-O-Methyltransferase
Vaccinia Capping
Enzyme
RNA polymerase + NTPs
GTP
SAM
3´
RNA transcript
5´
P
P
P
P
P
P
G
3´
Cap-1 mRNA
5´
P
P
P
m
7
G
2´-O-methylation
OCH
3
DNA template
RNA polymerase
promoter
5´
3´
3´
5´
+
3
continued on page 4...
TRANSCRIPTION FOR ENZYME-
BASED CAPPING (POST-
TRANSCRIPTIONAL CAPPING)
Standard RNA synthesis reactions produce
the highest yield of RNA transcript (typically
≥100 μg per 20 μl in a 1 hr reaction using the
HiScribe
™
Quick T7 High Yield RNA Synthesis
Kit, NEB #E2050S). Transcription reactions are
highly scalable, and can be performed using an
all-inclusive kit (e.g., HiScribe kits), or individual
reagents. More information on the HiScribe kits
can be found later in the article.
Following transcription, the RNA is treated with
DNase I (NEB #M0303) to remove the DNA
template, and purified using an appropriate
column, kit or magnetic beads, prior to capping.
This method produces high yields of RNA with
5´-triphosphate termini that must be converted
to cap structures. In the absence of template-
encoded poly(A) tails, transcripts produced using
this method bear 3´ termini that also must be
polyadenylated in a separate enzymatic step, as
described below in “Post-transcriptional capping
and Cap-1 methylation”.
TRANSCRIPTION
WITH DINUCLEOTIDE
CO-TRANSCRIPTIONAL CAPPING
In co-transcriptional capping, a cap analog is
introduced into the transcription reaction, along
with the four standard nucleotide triphosphates,
in an optimized ratio of cap analog to GTP 4:1.
This allows initiation of the transcript with the cap
structure in a large proportion of the synthesized
RNA molecules. This approach produces a mixture
of transcripts, of which ~80% are capped, and the
remainder have 5´-triphosphate ends. Decreased
overall yield of RNA products results from the
lower concentration of GTP in the reaction
(Figure4).
There are several cap analogs used in co-
transcriptional RNA capping (3,4). The most
common are the standard 7-methyl guanosine
(m7G) cap analog and anti-reverse cap analog
(ARCA), also known as 3´ O-me 7-meGpppG cap
analog. ARCA is methylated at the 3´ position
of the m7G, preventing RNA elongation by
phosphodiester bond formation at this position.
Thus, transcripts synthesized using ARCA contain
5´-m7G cap structures in the correct orientation,
with the 7-methylated G as the terminal
residue. In contrast, the m7G cap analog can be
incorporated in either the correct or the reverse
orientation.
HiScribe T7 ARCA mRNA Synthesis kits
(NEB #E2060 and #E2065) contain reagents,
including an optimized mix of ARCA and NTPs,
for streamlined reaction setup for synthesis of co-
transcriptionally capped RNAs.
TRANSCRIPTION WITH
CLEANCAP
®
REAGENT AG
CO-TRANSCRIPTIONAL CAPPING
The use of CleanCap reagent AG results
in significant advantages over traditional
dinucleotide co-transcriptional capping. CleanCap
Reagent AG is a trinucleotide with a 5´-m7G
joined by a 5´-5´ triphosphate linkage to an AG
sequence . The adenine has a methyl group on the
2´-O position (Figure 4). The incorporation of
this trinucleotide in the beginning of a transcript
results in a Cap-1 structure.
In order to use CleanCap Reagent AG in an in
vitro transcription reaction the template must
contain an AG in place of a GG following the T7
promoter in the initiation sequence.
Unlike traditional co-transcriptional capping,
reduction of GTP concentration is not required
and therefore yield is higher and high capping
eencies, >95%, are achieved (Figure 5).
TRANSCRIPTION WITH COMPLETE
SUBSTITUTION WITH MODIFIED
NUCLEOTIDES
RNA synthesis can be carried out with a
mixture of modified nucleotides in place of the
regular mixture of A, G, C and U triphosphates.
For expression applications, the modified
nucleotides of choice are the naturally occurring
5´-methylcytidine and/or pseudouridine in the
place of C and U, respectively. These have been
demonstrated to confer desirable properties to
the mRNA, such as increased mRNA stability,
increased translation, and reduced immune
response in the key applications of protein
replacement and stem-cell dierentiation (1). It
is important to note that nucleotide choice can
influence the overall yield of mRNA synthesis
reactions.
Fully substituted RNA synthesis can be achieved
using the HiScribe T7 mRNA Kit with CleanCap
Reagent AG (NEB #E2080), HiScribe T7 High-
Yield RNA Synthesis Kit (NEB #E2040) or
HiScribe SP6 RNA Synthesis Kit (NEB #E2070)
in conjunction with NTPs with the desired
modification. Transcripts made with complete
replacement of one or more nucleotides may be
post-transcriptionally capped (see next section), or
may be co-transcriptionally capped by including
CleanCap Reagent AG, ARCA or another cap
analog, as described previously.
If partial replacement of nucleotides is desired, the
HiScribe T7 ARCA mRNA Synthesis Kits (NEB
#E2060 and #E2065), may be used with added
modified NTPs, to produce co-transcriptionally
capped mRNAs, as described above. Alternatively,
the HiScribe T7 Quick RNA Synthesis Kit (NEB
#E2050) may be used to prepare transcripts for
post-transcriptional capping.
POST-TRANSCRIPTIONAL CAPPING
AND CAP-1 METHYLATION
Post-transcriptional capping is often
performed using the mRNA capping system
from Vaccinia virus. This enzyme complex
converts the 5´-triphosphate ends of in vitro
transcripts to m7G-cap (Cap-0) required for
ecient protein translation in eukaryotes. The
Vaccinia Capping System (NEB #M2080)
comprises three enzymatic activities (RNA
triphosphatase, guanylyltransferase, guanine
N7-methyltransferase) that are necessary for
the formation of the complete Cap-0 structure,
m7Gppp5´N, using GTP and the methyl
donor S-adenosylmethionine. As an added
option, the inclusion of the mRNA Cap 2´
O-Methyltransferase (NEB #M0366) in the
same reaction results in formation of the Cap-1
structure (m7Gppp5´Nm), a natural modification
in many eukaryotic mRNAs responsible for
evading cellular innate immune response against
foreign RNA. This enzyme-based capping
approach results in a high proportion of capped
message, and it is easily scalable. The resulting
capped RNA can be further modified by poly(A)
addition before final purification.
FIGURE 4:
Structure of CleanCap
Reagent AG
OPP
O
OO
O
O
O
H
2
N
HN
N
+
CH
3
CH
3
N
N
O
O
HO OH
OP
P
O
O
O
NH
2
N
N
N
N
OO
O
OO
3Na
+
O
OHHO
O
NH
2
N
N
N
NH
FIGURE 5:
Comparison of RNA yields
from in vitro transcription
reactions
All reactions were performed with 5 mM CTP, 5 mM UTP and 6 mM
ATP. Standard IVT reactions included 5 mM GTP and no cap analog.
ARCA reactions contained a 4:1 ratio of ARCA:GTP (4 mM:1 mM). IVT
with CleanCap Reagent AG contained 5 mM GTP and 4 mM CleanCap
Reagent AG and was performed as described below (Standard mRNA
Synthesis). Reactions were incubated for 2 hours at 37°C, purified and
quantified by NanoDrop
®
.
0
20
40
60
80
100
120
140
Standard
IVT
IVT with
ARCA
IVT with
CleanCap
®
Reagent AG
RNA Yield (µg)
Analysis of capped RNA function in transfected mammalian cells
(A) Schematic representation of reporter mRNA transfection workflow. (B) Expression of Cypridina luciferase (CLuc) after capping using different
methods. High activity from all capped RNAs is observed.
The effect of capping can be studied by delivering the mRNA to cultured mammalian cells and monitoring its translation.
Using RNA encoding secreted luciferases (e.g., Cypridina luciferase, CLuc) the translation can be monitored by assaying its
activity in the cell culture medium (Fig. A).
CLuc mRNA was synthesized and capped post-transcriptionally (Cap 0 or Cap 1) or co-transcriptionally (as described above)
using standard (7mG) or anti-reverse cap analog (ARCA). For consistency, the mRNAs were prepared from templates
encoding poly-A tails of the same length.
After capping, the mRNA was purified using magnetic beads and quantified before transfection into U2OS cells using
the TransIT
®
mRNA transfection reagent following the manufacturer’s protocol. CLuc activity was measured 16 hrs after
transfection using the BioLux
®
Cypridina Luciferase Assay Kit (NEB #E3309).
Virtually no luciferase reporter activity was observed in conditions where uncapped RNA was transfected (Fig. B). In contrast,
robust activity was detected from cells transfected with RNA capped using the methods described above. As anticipated,
lower activity was observed from cells transfected with mRNA capped using the 7mG cap analog as compared to ARCA-
capped mRNA.
RLU
0.0
1.0x10
7
1.5x10
7
2.0x10
7
5.0x10
6
Cap 0 Cap 1 7mG
Cap Structure
ARCA
Uncapped
Make mRNA Transfect cells Read light outputHarvest media
44
A-TAILING USING E. COLI
POLY(A) POLYMERASE
The poly(A) tail confers stability to the mRNA
and enhances translation eciency. The poly(A)
tail can be encoded in the DNA template by
FIGURE 6:
Analysis of capped and polyadenylated RNA
(A) Agilent
®
Bioanalyzer
®
analysis of capped and polyadenylated RNA. Longer tails are produced by increasing the enzyme
concentration in the reaction. Calculated A-tail lengths are indicated over each lane. Lanes: L: size marker,1: No poly-A tail, 2: 5 units, 3 :15
units, 4 : 25 units of E. coli Poly(A) Polymerase per 10 µg CLuc RNA in a 50 µl reaction. (B) Effect of enzymatic A-tailing on the luciferase
reporter activity of CLuc mRNA.
continued on page 5...
using an appropriately tailed PCR primer, or it
can be added to the RNA by enzymatic treatment
with E. coli Poly(A) Polymerase (NEB #M0276).
The length of the added tail can be adjusted by
titrating the Poly(A) Polymerase in the reaction
(Figure 6).
The importance of the A-tail is demonstrated by
transfection of untailed vs. tailed mRNA. When
luciferase activity from cells transfected with
equimolar amounts of tailed or untailed mRNAs
were compared, a significant enhancement of
translation eciency was evident (Figure 6).
HiScribe T7 ARCA mRNA Synthesis Kit (with
tailing) (NEB #E2060) includes E. coli Poly(A)
Polymerase, and enables a streamlined workflow
for the enzymatic tailing of co-transcriptionally
capped RNA.
For mRNA synthesis from templates with
encoded poly(A) tails, the HiScribe T7 ARCA
mRNA Synthesis Kit (NEB #E2065) provides
an optimized formulation for co-transcriptionally
capped transcripts.
SUMMARY
In summary, when choosing the right workflow
for your functional mRNA synthesis needs, you
must balance your experimental requirements for
the mRNA (e.g., internal modifed nucleotides)
with scalability (i.e., ease-of-reaction setup vs.
yield of fnal product).
In general, co-transcriptional capping of
mRNA with template encoded poly(A) tails
or post-transcriptional addition of poly(A) tail
is recommended for most applications. This
approach, using the HiScribe T7 mRNA Kits with
CleanCap Reagent AG (NEB #E2080), enables
the quick and streamlined production of one or
many transcripts with typical yields of ≥90 μg
per reaction, totaling ~1.8 mg per kit.
Post-transcriptional mRNA capping with Vaccinia
Capping System is well suited to larger scale
synthesis of one or a few mRNAs, and is readily
scalable to produce gram-scale quantities and
beyond. Reagents for in vitro synthesis of mRNA
are available in kit form or as separate components
to enable research and large-scale production.
Products from NEB are available for each step of
the RNA Synthesis Product Workflow. GMP-
grade products suitable for manufacture of large
scale manufacture of therapeutic mRNA are
available through our Customized Solution Group.
References:
1. Warren, L., et al. (2010) Cell Stem Cell, 7, 618-630.
2. Angel, M. and Yanik, M.F. (2010) PLoS One, 5:e11756.
3. Yakubov, E., et al. (2010) Biochem. Biophys. Res. Commun. 394, 189.
4. Geall, A.J., et al. (2012) Proc. Natl. Acad. Sci. USA, 109,
14604-14609.
5. Ramaswamy, S., et al. (2017) Proc. Natl. Acad. Sci. USA, 114,
E1941-E1950.
6. Ma, Y., et al. (2014) PLoS One, 9:e89413.
7. Ota, S., et al. (2014) Genes Cells, 19, 555-564.
8. Bassett, A. R., et al. (2013) Cell Rep. 4, 220–228.
9. Wells, S.E., et al. (1998) Molecular Cell 2, 135–140.
A.
B.
RLU
0.0
3.0x10
7
4.0x10
7
5.0x10
7
A B
1.0x10
7
2.0x10
7
0 10 20 30
PAP units
+97 +167 +183
2 3L
2,000
1,000
500
200
25
1 4
mRNA SYNTHESIS WORKFLOW
EXAMPLE & AVAILABLE NEB PRODUCTS
TEMPLATE
GENERATION
IN VITRO
TRANSCRIPTION
RNA
CAPPING
POLY(A)
TAILING
RNA
PURIFICATION
Q5
®
High-Fidelity DNA
Polymerase
HiScribe
®
T7 mRNA Kit with CleanCap
®
Reagent AG
Monarch
®
RNA
Cleanup Kit (10 µg)
HiScribe T7 ARCA mRNA Synthesis Kit (with tailing)
dNTP solution mixes HiScribe T7 ARCA mRNA Synthesis Kit
E. coli Poly(A)
Polymerase
Monarch RNA
Cleanup Kit (50 µg)
BspQI*
HiScribe T7
High Yield RNA
Synthesis Components
Vaccinia
Capping System
Monarch RNA
Cleanup Kit (500 µg)
DNA Assembly
•
NEBuilder HiFi DNA
Assembly
•
Golden Gate Assembly
HiScribe T7 Quick High
Yield RNA Synthesis Kit
mRNA Cap
2´-O-Methyltranferase
Lithium Chloride
HiScribe SP6 High Yield
RNA Synthesis Kit
ARCA and other
mRNA cap analogs
T3 & SP6 RNA Polymerases
T7 RNA Polymerase
Hi-T7 RNA Polymerase
Companion Products Companion Products
RNase inhibitor
(Murine)
Monarch RNA Cleanup
Binding Buffer
RNase Inhibitor
(Human Placental)
Monarch RNA Cleanup
Wash Buffer
Pyrophosphatase,
Inorganic (E. coli)
Nuclease-free Water
Pyrophosphatase,
Inorganic (Yeast)
DNase I (RNase-free)
NTPs
= available in GMP-grade
*
NEB can offer large-scale preparations of restriction enzymes using
Recombinant Albumin (BSA-free)
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®
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