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TripleMaster and Perfect gDNA Blood Mini Kit Team Up,,, to Amplify Long gDNA Templates

TripleMaster and Perfect gDNA Blood Mini Kit Team Up
to Amplify Long gDNA Templates

Jennifer Halcome
Eppendorf 5 Prime, Boulder, CO, USA Introduction

Eppendorf provides a complete system for long-range PCR1 analysis of gDNA. The Perfect gDNA Blood Mini Kit offers an easy and effective method for isolating high molecular weight gDNA. The resulting gDNA can be used in combination with the Eppendorf Mastercycler gradient thermocycler and Eppendorf TripleMaster PCR System to amplify long templates.

In the past, scientists needing pure, high molecular weight gDNA would have to purchase special products or use lengthy, highly involved methods for DNA isolation. The Perfect gDNA Blood Mini Kit provides a method of extraction resulting in DNA ranging in size from 20 kb to 90 kb. PCR fragments up to 24 kb in size have been amplified using gDNA purified by the Eppendorf method. The following set of experiments was designed to demonstrate how Perfect gDNA Blood Mini Kit and TripleMaster team up to enable easy amplification of long templates.

Materials and Methods

Pulse Field Gel Electrophoresis:
A number of samples isolated using the Perfect gDNA Blood Mini Kit (Eppendorf) and QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany) were analyzed using pulse field gel electrophoresis (see Fig. 1). Samples were loaded on a 1% agarose gel in 0.5X TBE Buffer using the BIO-RAD Pulse Field Electrophoresis apparatus (BIO-RAD, Hercules, CA, USA). A Low Range PFG Marker (NEB, Beverly, MA, USA) and Lambda Hind III Marker (Worthington, Lakewood, NJ, USA) were run with the samples for size determination. 250 ng of gDNA template were loaded onto the gel. The template was isolated from human whole blood stored under the following conditions: 4C (1-day), 4C (2 months), and -20C (1-day). The isolated gDNA was stored at 4C for 5 months prior to electrophoresis.

Long-Range PCR:
gDNA was isolated using the Perfect gDNA Blood Mini Kit (Eppendorf) and QIAamp DNA Blood Mini Kit (Qiagen). 22 kb and 24 kb fragments of human gDNA were amplified using the Eppendorf TripleMaster PCR System. The following components were used with the final concentrations presented in parentheses:

Universal forward primer for human tPA gene (400 nM)
Reverse primer for human tPA gene (400 nM)
10X Tuning Buffer (1X)
dNTP mix (500 M)
TripleMaster Enzyme Mix (0.04 U/l)

The amounts of template for the 22 kb and 24 kb fragment amplifications were 400 ng and 500 ng respectively. Both reactions were carried out simultaneously on the Eppendorf Mastercycler gradient using the following cycling parameters:

Cycling parameters
93C for 3 minutes
Then
10 cycles:
93C for 15 seconds
65C for 30 seconds
68C for 20 minutes
Then
8 cycles:
93C for 15 seconds
65C for 30 seconds
68C for 20 minutes
+ 20 seconds each cycle
Then
4C hold

Sample Analysis
Samples were analyzed by loading 30% of the 50 l reaction on a 0.6% agarose gel (see Fig. 2). The gel was run for 2 hours at 60 V, stained with ethidium bromide and photographed.

Results

Fig. 1 shows results from the pulse field gel electrophoresis experiment. Eppendorf samples average about 70 kb. It appears that blood and final sample storage conditions do not affect the overall size of the genomic DNA. The gDNA isolated using the Qiagen product averages between 23 and 48.5 kb. The presence of larger amounts of higher molecular weight gDNA in the Eppendorf samples provide a significant advantage for long-range PCR efforts. In the second experiment, using the Eppendorf TripleMaster PCR System, DNA is easily amplified in both the 22 kb and 24 kb PCR reactions. In fact, lanes with the Eppendorf-isolated gDNA are overloaded with product. The TripleMaster PCR System is able to ensure some amplification of the 22 kb fragment even with the lower molecular weight DNA isolated using the Qiagen product, but the 24 kb reaction generates no amplification product.

Fig. 1: Field inversion gel electrophoresis

Lane 1: Low Range PFG Marker Lane 2: Lambda Hind III Marker Lane 3: Eppendorf 4C, 1 day Lane 4: Qiagen 4C, 1 day Lane 5: Eppendorf 4C, 2 months Lane 6: Qiagen 4C, 2 months Lane 7: Eppendorf-20C, 1 day Lane 8: Qiagen 20C, 1 day Fig. 2: Long-range PCR

Lane 1: Lambda Hind III Marker Lanes 24: Eppendorf 22 kb Lanes 57: Qiagen 22 kb Lanes 810: Eppendorf 24 kb Lanes 1113: Qiagen 24 kb Lane 14: Lambda Hind III Marker Conclusions
As the first component of the team, the Perfect gDNA Blood Mini Kit provides the researcher with pure, high molecular weight gDNA from blood. The robustness of the TripleMaster PCR System provides the final component of the system allowing easy amplification of long templates. The TripleMaster/Perfect gDNA Blood Mini team is the first reliable miniprep system on the market for template preparation in combination with long-range PCR.

References

  1. Use of the Polymerase Chain Reaction (PCR) process to amplify nucleic acids is covered by U.S. Patent Numbers 4683105 and 4683202 owned by Roche Molecular and requires a license.

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