Storage And Stability
PCR Purification Kit can be stored for up to 2 years at room temperature (15-25°C) or at 4°C for storage periods longer than 12 months. Any precipitate in the buffers can be re-dissolved by incubating at 37°C before use.
The Eco-Friendly PCR Purification Kit is designed for rapid and efficient purification of DNA from PCR and other enzymatic reaction mixtures. The kit utilizes a proprietary silica-based membrane technology in the form of a convenient spin column, eliminating the need for tedious resin manipulations or toxic phenol-chloroform extractions. The PCR Purification Kit effectively removes primers, dNTPs, unincorporated labeled nucleotides, enzymes and salts from PCR and other reaction mixtures. Each purification column has a total binding capacity of up to 30μg of DNA and the entire procedure takes just 15 minutes. The purified DNA can be used in common downstream applications such as sequencing, restriction digestion, labeling, ligation, cloning, in vitro transcription, blotting or in situ hybridization.
A reaction mixture containing DNA is combined with the binding buffer and added to a purification column. A chaotropic agent in the binding buffer denatures proteins and promotes DNA binding to the silica membrane in the column. The chaotropic agent of regular PCR purification method usually contains guanidine salt, a hazardous protein denaturant. The Eco-Friendly system is the only method available that does not utilize Guanidine Salt, in the silica based purification system, which will ensure the safety and health of scientists and the environment.
Safety and environmental protection: No toxic chemicals in the solution, which is safer for people; No toxic chemicals in the waste, which is easy to dispose and good for environment.
High integrity: Protect the terminal base pairs of the DNA fragment.
Fast: Procedure takes 15 minutes.
Highly efficient: 85-100% recoveries.
Pure DNA: A260/A280= 1.7-1.9.
Fast and efficient purification of DNA fragments ideal for use in all conventional molecular biology procedures including:
ü conventional restriction digestion
ü in vitro transcription
The kit is tested in the purification of 50 bp, 1 kb and 10 kb PCR products according to the protocol. The quality of the purified DNA is evaluated spectrophotometrically, by agarose gel electrophoresis, digestion with restriction enzymes and automated fluorescent sequencing.
? Prior to the initial use of the kit, dilute the Wash Buffer(PE) with ethanol (100%):
15 ml ×2
20 ml ×2
60 ml ×2
80 ml ×2
75 ml ×2
100 ml ×2
After the ethanol has been added, mark the check box on the bottle to indicate the completed step.
? Freshly prepared electrophoresis buffers should be used both for gel preparation and for gel running.
? All purification steps should be carried out at room temperature.
1.Add a 1 volume of Solution F and 1 volume of 96%-100% ethanol to 1 volume of completed PCR mixture (e.g. for every 100 μl of reaction mixture, add 100 μl of Solution F and 100 μl of ethanol). Mix thoroughly.
2.Transfer up to 800 μl of the solution from step 1 to the spin column. Incubate for 2 minutes.
3.Centrifuge for 1 min at 12,000 rpm. Discard the flow-through.
Note. If the total volume exceeds 800 μl, the solution can be added to the column in stages. After the addition of 800 μl of solution, centrifuge the column for 30-60 s and discard flow-though. Repeat until the entire solution has been added to the column membrane.
4.Add 500 μl of Solution PE (diluted with the ethanol) to the column. Centrifuge for 1 min at 12,000 rpm. Discard the flow-through and place the purification column back into the collection tube. Repeat this step again.
5.Centrifuge the empty column for an additional 3 min to completely remove any residual wash buffer.
Note. This step is essential as the presence of residual ethanol in the DNA sample may inhibit subsequent reactions.
6.Transfer the column to a clean 1.5 ml microcentrifuge tube (not included). Add 30-100 μl of Eluent Buffer (prewarm to 65℃)to the center of the column membrane and incubate for 2 min. Centrifuge for 1 min at 12,000 rpm. Discard the column and store the purified DNA at -20℃.
Note? For low DNA amounts the elution volumes can be reduced to increase DNA concentration. An elution volume between 20-50 μl does not significantly reduce the DNA yield. However, elution volumes less than 10 μl are not recommended.