GB-Clean™ PCR/DNA 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. The kit can be used for purification of DNA fragments from 50 bp to 40 kb with recovery rates up to 100%. Each purification column has a total binding capacity of up to 30μg of DNA and the entire procedure takes just 15 minuted. 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.
Principle
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. As an added convenience, the binding buffer contains a color indicator that allows for easy monitoring of the solution pH for optimal DNA binding. Impurities are removed with a simple wash step. Purified DNA is then eluted from the column with the Elution Buffer. The recovered DNA is ready for use in downstream applications.
Content: Solution BD, Solution PE, Eluent Buffer, Spin Columns
Storage And Stability
PCR Purification Kit can be stored for up to 12 months 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.
Features
Highly efficient: 85-100% recoveries in the range of 50 bp-40 kb.
Convenient: Spin columns are capped and assembled with collection tubes.
Fast: Procedure takes 15 minutes.
Pure DNA: A260/A280= 1.7-1.9.
Applications
Fast and efficient purification of DNA fragments ideal for use in all conventional molecular biology procedures including:
• conventional restriction digestion
• automated fluorescent or radioactive sequencing
• PCR
• in vitro transcription
Quality Control
The kit is tested in the purification of 50 bp and 1 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.
Important Notes
• Prior to the initial use of the kit, dilute the Wash Buffer(PE) with ethanol (100%):
|
N1091(50preps) |
N1092(100preps) |
N1093(200preps) |
|
|
Wash Buffer(PE) |
15 ml |
15 ml ×2 |
20 ml ×2 |
|
Ethanol |
60 ml |
60 ml ×2 |
80 ml ×2 |
|
Total Volume |
75 ml |
75 ml ×2 |
100 ml ×2 |
After the ethanol has been added, mark the check box on the bottle to indicate the completed step.
• Examine the Gel Solubilization Buffer (BD) for precipitates before each use. Re-dissolve any precipitate by warming the solution to 37°C and cooling to 25°C.
• 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.
Protocol
1.Add a 1:1 volume of Solution BD to completed PCR mixture (e.g. for every 100 μl of reaction mixture, add 100 μl of Binding Buffer). Mix thoroughly.
2.Transfer up to 800 μl of the solution from step 1 to the spin column. Incubate for 2 minutes. 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.
3.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.
4.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.
5.Transfer the column to a clean 1.5 ml microcentrifuge tube (not included). Add 30-100 μl of Elution Buffer (prewarm to 65℃)to the center of the column membrane and incubate for 2 minutes. 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.
