2×PCR Mix

2×PCR Mix

Composition

Storage and Expiry date

- The validity period is more than two years when stored at 20 ℃; the validity period is 6 months when stored at 2~8 ℃ . Repeated freezing and thawing does not affect use.

Product Description

2×PCR Mix is an optimized double-concentration PCR master mix. Suitable for conventional PCR , it can amplify fragments up to 4 kb from complex genomic DNA or up to 5 kb from lambda DNA . PCR enhancers and protein stabilizers synergistically improve PCR efficiency and sensitivity, making them ideal for low-copy template amplification. The product is easy to use. You only need to take 0.5 times the volume of the PCR system of 2×PCR Mix , add primers and templates, and make up the volume with ddH 2 O.

The target product amplified by this kit has an A base attached to its 3' end and can be directly cloned into T-Vector . The product contains a blue electrophoresis indicator dye, and the amplified product can be directly detected by agarose electrophoresis.

PCR system components

1．  template DNA : Many residual nucleic acid extraction reagents will affect the PCR reaction, including proteases, protein denaturants ( such as SDS , guanidine salts ) , high-concentration salts (KAc , NaAc , sodium octanoate, etc. ) and high-concentration EDTA , etc. The amount of low-purity template (such as template obtained by boiling method) should not exceed 1/10 of the PCR reaction system (for example, the volume of template added to a 50 µl reaction system should not exceed 5 µl ). If the purity of the template DNA is too poor, you can use the Simgen DNA Purification Kit ( Cat. No. 2101050 ) to purify and concentrate the template DNA . The amount of template purified by Simgen DNA Purification Kit can be up to 1/2 of the volume of the PCR reaction system .

2．  template DNA : A very small amount of DNA can also be used as a PCR template, but to ensure the stability of the reaction, it is recommended to use 10 ^{4 copies of the target sequence as a template for the} 50 µl system . Recommended usage amount of template DNA :

Human genomic DNA :  50 ng~500 ng/50 µl PCR reaction system

E. coli genomic DNA :  10 ng~100 ng/50 µl PCR reaction system

λDNA :                        0.5 ng~5 ng/50 µl PCR reaction system

Plasmid DNA :             0.1 ng~10 ng/50 µl PCR reaction system

If you need to use the amplification product as a template for further amplification, you should dilute the amplification product at least 1,000 to 10,000 times before using it as a template.

3．  Primer concentration: Generally, the concentration of each primer is 10 µM (50×) , and the working concentration is 0.2 µM . Excessive primers may cause non-specific amplification, while too few primers may reduce amplification efficiency.

PCR parameter settings

1.       Pre-denaturation: Generally, pre-denaturation is 94 °C, 1~5 minutes . If the denaturation temperature is too high or the time is too long, Taq enzyme activity will be lost.

2.       Annealing: Annealing temperature is the key to PCR . Too high a temperature may reduce the yield, and too low a temperature may produce primer dimers or non-specific amplification. When trying PCR amplification for the first time, it is recommended to directly use 55 °C as the annealing temperature, or try to use a temperature 5 °C lower than the Tm ( if the Tm of the two primers are different, refer to the lower Tm) as the annealing temperature. Generally, primer synthesis companies will provide the Tm of the synthesized primers, and the primer Tm can also be estimated according to this formula : Tm = 2 ℃ × (A+T) + 4 ℃ × (G+C) . The optimal annealing temperature needs to be determined by gradient PCR .

3.       Extension: The extension temperature is usually 72 °C . The extension time depends on the length of the target DNA fragment. The required extension is calculated at 1 kb/min .time, too long may result in non-specific increases. After the cycle is completed, continue extending for 5 to 10 minutes to obtain a complete double-stranded product.

4.       Number of cycles: Generally, 25 to 35 cycles are used. For low-copy templates, the number of cycles can be increased appropriately. Excessive cycle numbers may increase nonspecific amplification and reduce specific products.

Steps:

1.        Thaw 2×PCR Mix , ddH 2 O , template DNA and primers at room temperature and place on ice.

2.        After thawing, turn each component upside down and mix evenly. Prepare the PCR reaction solution according to the following composition:

* Note: The above examples are the components added to a 50 µl reaction system. If other volumes of reaction systems are needed, please increase or decrease the components in proportion.

3.        Flick the PCR reaction tube with your finger to mix thoroughly and centrifuge briefly.

4.        PCR reaction cycle setup example

94 ℃ 3 minutes

94 ℃ 30 sec ]

※ 55 ℃ 30 sec > 35 Cycles

§      72 ℃ 1 min

72 ℃ 5 minutes

※Subject to the actual optimal annealing temperature.

§     Calculated at 1 kb/ min .

For amplification of target fragments below 300 bp , a two-step amplification method can be used to save amplification time:

94 ℃ 3 minutes

94 ℃ 20 sec ]

     35 Cycles

60 ℃ 1 min

72 ℃ 5 minutes

5.        Result detection: Take 5-10 µl amplification product for direct agarose electrophoresis detection.

* The relationship between agarose gel concentration and the optimal resolution range of linear DNA :