peqGOLD Cycle‐Pure Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
The amount of agarose excised from the gel should be as small as possible.	Remove the flow-through by aspiration. Avoid contamination of the collection tube rim.

Upstream tips
The amount of agarose excised from the gel should be as small as possible.
Protocol tips
Remove the flow-through by aspiration. Avoid contamination of the collection tube rim.

Publication protocol

Materials and methods
The present analyses were carried out using 62 specimens of 28 different mosquito species of 10 genera. The method was applied to mosquitoes which had been stored at −20, −80 °C or room temperature, or had been preserved in 70% ethanol or had been stored as dry material for between 1 month and 3 years. Different amounts of tissue were used to test the minimum amount of tissue required to produce positive PCR results in the gel electrophoresis. Barcoding with direct PCR was carried out with the Phusion® Blood Direct PCR Kit (Thermo Fisher Scientific, Inc., Waltham, MA, U.S.A.). The reaction mixture (50 µL) consisted of 25 µL of direct PCR buffer, 1 µm of each primer and 1 U Phusion® II DNA polymerase. Millipore water was added to a total volume of 50 µL, as were different tissue samples (not homogenized) as DNA template (Table 1). The Phusion® high‐fidelity polymerase provided in the kit has a 50‐fold lower error rate than commercial standard Taq polymerases and is extremely resistant to inhibitors found in samples that are not extracted or purified before amplification [see #F‐547S (Thermo Fisher Scientific, Inc., Waltham, MA, U.S.A.) and #M0530S (New England Biolabs, Inc., Beverly, MA, U.S.A.)]. The amplification of a cytochrome c oxidase subunit 1 (COX1) gene fragment was performed in a thermocycler (Eppendorf Vertrieb Deutschland GmbH, Hamburg, Germany) using BC‐Kumar forward/reverse primers (Kumar et al., 2007). The parameters for PCR cycling were as follows: one cycle of 94 °C for 2 min; 40 cycles of 94 °C for 60 s, 59 °C for 60 s and 72 °C for 60 s, followed by a terminal extension of 72 °C for 5 min and a final ramping to 8 °C. The quality and yield of DNA were analysed by Midori Green (Nippon Genetic Europe GmbH, Düren, Germany) staining and agarose gel electrophoresis. Positive samples were purified using the peqGOLD Cycle‐Pure Kit (Peqlab Biotechnology GmbH, Erlangen, Germany) and Illustra™ ExoStar™ 1‐Step (GE Healthcare, Chalfont St Giles, U.K.). The subsequent Sanger sequencing was performed using Seqlab (Seqlab‐Sequence Laboratories Göttingen GmbH, Göttingen, Germany) and Microsynth (Microsynth AG, Balgach, Switzerland) using the BC‐Kumar forward and/or reverse primer. Each sequence was edited using BioEdit (Hall, 1999) and compared with sequences deposited in GenBank using the blast algorithm (Altschul et al., 1997).

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Manufacturer protocol

Download the product protocol from VWR for peqGOLD Cycle‐Pure Kit below.

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