( rs were used. Sequencing ...)Eppendorf Perfectprep Gel Cleanup Kit

rs were used.

Sequencing
Sequencing was performed on 5 l of purified PCR products in triplicate. and Brand Q's Gel Extraction Kit were used for extraction and purification of the DNA fragment. Sequencing was carried out on a 377 ABI Sequencer. The results were analyzed and Phred Q20 scores were determined.

Labeling and hybridization
Dot Blot analysis was used to determine the effectiveness of labeling and hybridizing a DNA fragment purified using the Eppendorf Perfectprep Gel Cleanup Kit. The PCR product was gel extracted and purified, then labeled using the Ambion (Austin, TX, USA) Psoralen-Biotin Labeling Kit. 20 to 0.002 ng of gel extracted PCR product was blotted onto positively charged nylon and probed with the Psoralen-Biotin labeled probe. The Ambion Brightstar BioDetect Kit was used for detection of the labeled probe.

PCR Results

Amplification worked robustly when using DNA purified from the Eppendorf Perfectprep Gel Cleanup Kit and Brand Q (Fig. 2). PCR is a sensitive downstream application that can be affected by contaminants resulting from gel extraction methods. Based on the results obtained there is no detectable inhibition of the amplification reaction.

Figure 2: Amplification of gel extracted PCR product Lane 1 1 kb Marker Lanes 24 Brand Q Lanes 57 Eppendorf Lane 8 No Template Control Lane 9 1 kb Marker
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