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Catalog number selected: A This product is available through the Promega Helix onsite stocking program. We offer numerous convenient solutions to meet your lab's needs. Amplification efficiency and specificity can be improved by adjusting the annealing temperature according to the primer's T m or by performing two-step PCR. Three-step PCR includes denaturation, annealing, and extension steps. With this protocol, the annealing temperature should not exceed the extension temperature.
This lower extension temperature dramatically improves yields of longer amplification products by reducing the depurination rate that influences amplification. The optimal amount of template required depends on the complexity of the template and the copy number of the target sequence.
Approximately 10 4 copies of the target DNA sequence are required to detect the amplification product in 25—30 PCR cycles. It is important to note that not all polymerases can tolerate excessive amounts of template. DNA integrity is critical for amplification of long targets. DNA damage can also occur in acidic conditions; therefore, avoid using water for resuspending DNA templates. DNA is most stable at pH 7—8 or in buffered solutions.
The GC ratio varies across the genome. GC-rich regions of the genome are mostly concentrated in regulatory regions, including promoters, enhancers, and cis-regulatory elements. GC-rich tracts tend to form inverted repeats, or hairpin structures, that may not melt during the annealing step of PCR.
Therefore, amplification of GC-rich templates is hindered by inefficient separation of the two DNA strands. This results in truncated amplicons due to premature termination of polymerase extension. Use a polymerase optimized for amplification of GC-rich sequences. To find an enzyme, visit our selection guide. The recommended concentration of DMSO is between 2. Some templates may have long AT-rich stretches that are hard to amplify under standard reaction conditions.
The advantage of having AT-rich templates is that a lower extension temperature can be used. DNA replication at this reduced temperature appears to be reliable Su et al. Su, X. Nucl Acids Res. Magnesium is a required cofactor for thermostable DNA polymerases and is important for successful amplification. Salt neutralizes the negative charges on the phosphate backbone of DNA, stabilizing double-stranded DNA by offsetting negative charges that would otherwise repel one another.
To improve amplification of DNA fragments, especially fragments between and 1, bp, a KCl concentration of 70— mM is recommended. For amplification of longer products, a lower salt concentration appears to be more effective, whereas amplification of shorter products occurs optimally with higher salt concentrations.
This effect is likely because high salt concentration preferentially permits denaturation of short DNA molecules over long DNA molecules. This product and its components are not considered hazardous in their given concentrations. However, as with all scientific reagents this product should be handled and stored with care as standard practice.
Wear gloves. Care should be taken to avoid contact with skin or eyes. In case of contact with skin or eyes, wash immediately with water. A standard final concentration is 1. This will result in a working concentration of 5 mM MgCl 2. To increase the concentration of each PCR by 0. A suggested gradient is shown in the table below.
Pipette the required droplet volume onto the inside of each PCR tube to avoid touching the Master Mix and avoiding back transfer of reagents into the stock. Do the same for the 5 mM MgCl 2. Then close each PCR tube and flick to mix, ensuring the reaction mix is at the bottom of the tube.
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