Lars E. Peters, Walter Gross, and Jennifer Diers
Eppendorf 5 Prime, Inc., Boulder, CO, USA This experiment was performed to compare the Eppendorf TripleMaster PCR System with the long-range PCR products of leading competitors. Eppendorf was compared to competitors T and R in genomic DNA amplification; and competitors T, R, and AB in Lambda DNA amplification. Very stringent cycling conditions were used for all products in terms of a short extension time and low number of cycles. Please note: The cycling conditions are not according to the manufacturers instructions. These conditions prove the high processivity of the TripleMaster polymerase when tested against competitor products. In order to gain the success seen with the TripleMaster system, usual cycling conditions for competitors products would require much longer extension times and many more cycles.

Experimental Setup

Genomic DNA amplification

Reactions were executed using 200 ng of commercially purchased human gDNA (Roche). Primers targeting a 24 kb region of the human tPA gene were used. Primer sequences for the 24 kb human tPA universal forward and the 24 kb reverse primers may be found in the Eppendorf TripleMaster PCR System manual. Reaction components from each manufacturer were used as follows in the concentrations presented in parentheses:

Eppendorf TripleMaster PCR System

10X Tuning Buffer (1X)
Eppendorf dNTP Mix (0.5 mM)
MgCl2 (2.4 mM)
TripleMaster (2.0 U) Competitor R 5X Reaction Buffer (1X)
Eppendorf dNTP Mix (0.5 mM)
MgCl2 (2.4 mM)
Competitor R Polymerase (2.0 U) Competitor T Samples were processed with and without DMSO
10X Reaction Buffer (1X)
Eppendorf dNTP Mix (0.5 mM)
MgCl2 (2.4 mM)
Competitor T Polymerase (2.0 U)
DMSO (2%) Fig1: 24 kb target (tPA gen) amplification from human genomic DNA Lanes 12: Eppendorf TripleMaster PCR System Lanes 34: Competitor R Lanes 56: Competitor T Lanes 78: Competitor T +2% DMSO Lane M: Lambda Hind III Marker 40 kb target amplification from Lambda DNA Lanes 1011: Eppendorf TripleMaster PCR System Lanes 1213: Competitor R Lanes 14-15: Competitor T Lanes 1617: Competitor AB Lane M: Lambda Hind III Marker Cycling parameters

Initial denaturation
93C 3 min

10 cycles:
93C 15 sec
68C 27 min

17 cycles:
93C 15 sec
68C 21 min
+ 20 sec each cycle

Lambda DNA amplification

20 ng template was used for each reaction. Primers targeting a 40 kb region of the Lambda DNA were used for all amplification reactions. Reaction components from each manufacturer were used as follows in the concentrations presented in parentheses:

Eppendorf TripleMaster PCR System

10X Tuning Buffer (1X)
Eppendorf dNTP Mix (0.5 mM)
MgCl2 (2.4 mM)
TripleMaster (2.0 U) Competitor R 5X Reaction Buffer (1X)
Eppendorf dNTP Mix (0.5 mM)
MgCl2 (2.4 mM)
Competitor R Polymerase (2.0 U) Competitor T 10X Reaction Buffer (1X)
Eppendorf dNTP Mix (0.5 mM)
MgCl2 (2.4 mM)
Competitor T Polymerase (2.0 U) Competitor AB 3.3X Reaction Buffer (1X)
Eppendorf dNTP Mix (0.5 mM)
MgCl2 (2.2 mM)
Competitor AB Polymerase (2.0 U) Cycling parameters

Initial denaturation
93C 3 min

10 cycles:
93C 15 sec
68C 21 min

8 cycles:
93C 15 sec
68C 27 min
+ 20 sec each cycle

Sample analysis

Of each sample 25% was loaded onto a 0.4% agarose gel. The gel was stained with ethidium bromide and photographed.

Results

The Eppendorf samples clearly outperform the competition. In both the amplification of genomic DNA and Lambda DNA, the Eppendorf polymerase provides consistently high yields. Although competitor R samples perform in both cases, the Eppendorf yields are higher and more consistent. Slight amplification, however minimal, can be seen in competitor T samples. The addition of DMSO does not appear to increase amplification considerably. Competitor AB samples perform poorly. Overall the Eppendorf samples are superior to all competitor products tested.

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