Forum 10-3 nl_web
9/5/03
1:45 PM
Page 8
EPICENTRE Forum
Transcribe Milligram Amounts of RNA in as Little as 30 Minutes Using
the AmpliScribe™ T7-Flash™ Transcription Kit
The AmpliScribe™ T7-Flash™ Transcription
Kit, EPICENTRE’s new T7 in vitro transcription kit, produces more RNA in 30
minutes than other in vitro transcription
systems do in 2 hours.1 The standard
20-µl AmpliScribe T7-Flash transcription
reaction produces 160 to 180 µg (8 to
9 mg/ml) of a 1.4-kb RNA transcript in 30
minutes from 1 µg (0.05 µg/µl) of the control DNA template. Here we describe
methods to produce milligram quantities
of RNA from an AmpliScribe T7-Flash transcription reaction, even when the amount
of DNA template is limited.
Scale-up an AmpliScribe T7-Flash reaction to
make milligram amounts of RNA
Linear scale-up of all AmpliScribe T7Flash reaction components, including the
DNA template, yields milligram amounts
of RNA in 30 minutes (Figure 1). For
example, scaling up the standard 20-µl
reaction by 6-fold, to 120 µl (including
6 µg of the control DNA template), produced more than 1 mg of a 1.4-kb RNA
transcript in 30 minutes at 37ºC.
Transcribe more than 1 mg of RNA even from
limiting amounts of DNA template
For some in vitro transcription applications, the amount of template DNA is
limited. To determine whether the
AmpliScribe T7-Flash Kit could produce
milligram amounts of RNA from limited
amounts of DNA template, experiments
were performed in which the amount of
DNA template was held constant at 1 µg,
but all other reaction components were
scaled-up 8-fold, to 160 µl. As shown in
Figure 2, the reaction produced more than
a milligram of the 1.4-kb RNA transcript in
2 hours from 1 µg (6.25 x 10-3 µg/µl) of
DNA template. Using only 100 ng (6.25 x
10-4 µg/µl) of DNA template, an overnight
(16-hour) reaction yielded more than 1 mg
of the 1.4-kb RNA (>10,000-fold increase
in RNA yield). Increasing the reaction temperature from 37°C to 42ºC decreased the
time required to produce more than 1 mg
of RNA by at least 1 hour (data not shown).
Using only 10 ng (5.0 x 10-5 µg/µl) of
DNA template, a 10-fold scale-up reaction performed overnight at 37ºC yielded
more than 230 µg of the 1.4-kb RNA
transcript. This represents an impressive
23,000-fold increase in RNA yield compared to template concentration. The
same reaction performed overnight at
42ºC produced more than 300 µg of RNA.
By simply scaling up all reaction components, an AmpliScribe T7-Flash transcription
reaction can produce milligrams of an RNA
transcript in 30 minutes. Even with limited
amounts of DNA template, scaled-up reactions can generate milligrams of RNA by
extending the reaction time and increasing
the reaction temperature to 42°C.
Reference
1. Meis, R. and Pease, J. (2003) EPICENTRE
Forum 10(2), 6.
Figure 1. Milligram amounts
of RNA can be produced in
30 minutes by scaling up an
AmpliScribe™ T7-Flash™ transcription reaction. The standard
20-µl AmpliScribe T7-Flash reaction (1X) uses 1 µg of DNA
template to produce more than
160 µg of a 1.4-kb RNA transcript from the control DNA
template. By scaling up all reaction components, including the
DNA template, 6-fold or more,
reactions produce milligram
quantities of the 1.4-kb transcript in 30 minutes.
www.epicentre.com/flash.asp
AmpliScribe™ T7-Flash™
Transcription Kit
ASF3257
25 Reactions
ASF3507
50 Reactions
New!
AmpliScribe™ T3-Flash™
Transcription Kit
Figure 2. Milligram amounts of
RNA are produced from as little
as 100 ng of DNA template in
an AmpliScribe™ T7-Flash™
transcription reaction. All
components, except the DNA
template, were scaled-up 8-fold
to a final reaction volume of
160 µl. For all reactions, the
DNA template was maintained
at either 1 µg (purple bars) or
100 ng (maroon bars). Reactions
were performed at 37ºC for the
indicated length of time and
the yield of the 1.4-kb RNA
transcript was determined.
8
ASF03725
25 Reactions
ASF03750
50 Reactions
Contents:
AmpliScribe™ T7-Flash™ or T3-Flash™
Enzyme Solution (with RNase inhibitor)
AmpliScribe™ T7-Flash™ 10X Reaction Buffer
100mM ATP, CTP, GTP, UTP Solutions
RNase-Free DNase I
DTT
RNase-Free Water
Control DNA Template (linearized)
www.epicentre.com