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

ation. 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 el
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