At the end of the PCR reaction, it is essential to remove oligonucleotide primers, primer dimers and dNTP from the amplified DNA product. This experiment is based on the "Guide to Molecular Cloning, Third Edition", translated by Peitang Huang et al.
Operation method
Removal of oligonucleotides and excess dNTP from DNA amplification products by ultrafiltration
Principle
At the end of the PCR reaction, it is essential to remove the oligonucleotide primers, primer dimers and dNTP from the amplified DNA product. First, dNTP residues in the target product of DNA amplification fill in the sticky ends of the DNA product that has been cleaved by restriction endonucleases in the presence of residual heat-stabilizing DNA polymerase, making the latter product difficult to be further cloned. Secondly, excessive oligonucleotide primers make it less efficient to use the DNA product from the first PCR amplification as a template for the second PCR amplification reaction. Finally, the numerous restriction endonuclease sites within the primer dimer can compete with those at the ends of the amplified DNA fragments for the restriction endonuclease, thus affecting the efficiency of thinning of the amplified DNA fragments.
Materials and Instruments
Amplification Products Move I. Materials For more product details, please visit Aladdin Scientific website.
Chloroform Ethanol Sodium acetate TE
Preparative centrifuge or minicentrifuge Concentrator
1. Buffers and solutions
Chloroform
Ethanol
Sodium acetate (3 mol/L)
TE ( pH 8.0)
2. Nucleic acids and oligonucleotides
Amplification reaction products (20~200 μl)
3. centrifuge and rotor head
Preparative centrifuge or minicentrifuge
4. Specialized equipment
Concentrator (Centricon-100 or Microcon-100, Amicon)
II. Methods
1. Place 2 ml of TE (pH 8.0) in the reservoir of the Cemricon-100 concentrator. Carefully withdraw the amplification reaction product from underneath the upper layer of mineral oil with a pipette, or use chloroform to draw the sample from the reaction tube.
2. Place the transfer amplification reaction product into the reservoir of the Centricon-100 concentrator. Concentrator onto a suitable rotary head of a preparative centrifuge (e.g., a fixed right-angle rotary head). Insert this micro-concentrator onto the centrifuge using a filter cup with the translucent portion of the filter cup facing the bottom of the rotary head. Use another concentrator filled with an equal amount of liquid or a standard equilibrium tube as the corresponding equilibrium.
3. Centrifuge the concentrator for 30 min at 1000 g centrifugal force at a temperature of 4°C to 25°C after sampling.
4. Remove the concentrator from the centrifuge, discard the liquid in the translucent filter cup, convert the concentrator, and return it to the centrifuge (i.e., the concentrator's driven retention tube should now be placed toward the bottom of the rotary head ). Centrifuge again at 300 to 1000 g for 2 min.
5. Remove the concentrator from the centrifuge; remove the concentrate retention liquid and discard the remaining liquid in the concentrator unit. Transfer the concentrate from the concentrate retention tube to a new centrifuge tube.
6. If necessary, the sample in the retention concentrate tube may be precipitated with 1/10 times the volume of 3 mol/L sodium acetate and 2 to 3 times the volume of ethanol. This centrifuged dialyzed sample can be used for further operations (e.g. DNA sequencing and ligation reactions). One-step centrifugal dialysis with the Centricon-100 Filter Concentrator removes 95% of the residual primers and oligonucleotides from PCR-amplified DNA samples, and also inactivates the heat-stable DNA polymerase (presumably due to adsorption on the filter membrane). A single step of purification is usually sufficient for subsequent molecular manipulation of the amplified DNA. If necessary, trace amounts of oligonucleotide primers can be removed in a second 30 min centrifugal dialysis step. At step 4 above, empty the translucent filter cup, reassemble the concentrator, add 2 ml TE (pH 8.0) to the reservoir, and repeat steps 2 through 4.
The Cemricoti-100 Filter Concentrator can also be used for rapid purification of amplification products on a smaller scale. The general procedure is similar to the Centricon-100 Concentrator, except that in step 1, 400 μl of TE is added to the reservoir, followed by 20-100 μl of PCR amplification sample.
Centrifuge for 5-7 min at 3000 g in a variable speed centrifuge. The filtrate is removed and the concentrate sample is transferred to a retention cup and centrifuged again for 1 min at 300-1000 g. This procedure removes approximately 90% of the oligonucleotide primers and deoxyribonucleotides, and can be repeated to further purify the amplified DNA sample.