Product Introduction
2 × Rapid Taq Master Mix contains Taq DNA Polymerase, elongation promoting factor, dNTP and an optimized buffer system. The amplification speed of this product can reach 15 sec/kb, which is suitable for rapid PCR. The maximum amplification speed within 1 kb can reach 1 sec/kb, greatly saving reaction time. The pre-prepared 2 × Master Mix only needs to add primers and templates to perform amplification, which reduces pipetting operations and improves detection throughput and results reproducibility. The kit has excellent amplification performance and high storage stability. It is suitable for PCR amplification within 5 kb using genome as template and PCR amplification within 10 kb using plasmid and λDNA as template. The protective agent added to the system allows 2 × Master Mix to maintain stable activity after repeated freezing and thawing. The kit provides a version containing electrophoresis buffer and green loading dye, which can be directly electrophoresed after the reaction that is convenient to use. The PCR product has an adenine at the 3' end, which can be directly cloned into T vectors and is suitable for ClonExpress and TOPO cloning kits (GeneBio #C112/C113/C115/C601).
Product Advantages
Fast amplification speed: 15 sec/kb, the limit amplification speed can reach 1 sec/kb within 1 kb;
Convenient operation: amplification can be carried out by adding primers and templates, no operation on ice is required, and the product containing dye can be directly subjected to electrophoresis;
Good stability: repeated freezing and thawing for 50 times, no significant decrease in activity
Components
Storage
Store at -20℃ for up to 18 months; can be stored at 4℃ for 4 months after thawing
Citation
Huang, Jianfa, et al. "Migration of pre-induced human peripheral blood mononuclear cells from the transplanted to contralateral eye in mice." Stem cell research & therapy 12.1 (2021): 1-13.
Wang, Tianren, et al. "MFN2 Deficiency Impairs Mitochondrial Functions and PPAR Pathway During Spermatogenesis and Meiosis in Mice." Frontiers in cell and developmental biology 10 (2022).
Cao, Qiqi, et al. "The Recurrent Mutation in PATL2 Inhibits Its Degradation Thus Causing Female Infertility Characterized by Oocyte Maturation Defect Through Regulation of the Mos-MAPK Pathway." Frontiers in cell and developmental biology (2021): 116.
FAQ
Q1: The amplification efficiency is low and there are no amplified bands in the test group.
A1: (1) Primers
Check whether the synthetic primers have degraded due to improper storage; review primer design and use BLAST to examine primer specificity or redesign primers.
(2) Template
Long-term storage or repeated freeze-thaw cycles will lead to DNA breakage, nicking, or degradation, so the template should be the freshly prepared double-stranded DNA. An excessively high GC content in the template will make it difficult to separate DNA double strands; in this case, add PCR Enhancer (Cat. No. P021) to effectively lower the melting temperature. Crude sample templates may contain inhibitors, so it is recommended to lower the template concentration (use after dilution; if a plant leaf is used, make sure the plant is not rich in polysaccharides or polyphenols, take a fresh, young leaf, and trim it to the size of the end of a yellow pipette tip). If cDNA is used, check the purity and integrity of the RNA used for reverse transcription.
(3) Enzyme
The enzyme used in the reaction has been inactivated. Repeat the experiment with a fresh enzyme or an enzyme from another source.
(4) Amplification system
The reaction system has been prepared incorrectly. Repeat the experiment with the correct system.
(5) Reaction program
Check whether the denaturation temperature is correct and whether the temperature displayed on the PCR system is the same as the actual temperature. If the temperature is too high, the enzyme will be rapidly inactivated in the first few cycles. If the temperature is too low, the template will not be completely denatured. If yeast is used as the template, the pre-denaturation time may be extended to 10 minutes. If the annealing temperature is inappropriate, determine the appropriate annealing temperature by testing a gradient of annealing temperatures. If the target fragment is long, try the touchdown PCR program. Check whether the extension time is sufficient.
Q2: The amplified bands are faint.
A2: (1) Primers
Check whether the synthetic primers have degraded.
(2) Template
Check the quality of the template. Long-term storage or repeated freeze-thaw cycles will lead to DNA breakage, nicking, or degradation, so the freshly prepared double-stranded DNA should be used as the template. If the template has been used up, use serial dilutions of the initial amplification product as the templates for subsequent amplification.
(3) Reaction program
Try the touchdown PCR program; increase the extension time and the number of cycles.
Q3: Amplification specificity is poor, or there are non-specific products.
A3: (1) Primers
Primer design is not optimal. If primers have bound to non-specific sites in the target sequence or to each other to form dimers, optimize by reducing the primer concentration or redesigning primers if necessary.
(2) Template
If the template is impure or contaminated, prepare a new template.
If the template has degraded, or too much template has been used, examine the integrity and concentration of the template by electrophoresis, and purify the template again if necessary. Consult the Instructions for Use for the template input amount.
(3) Reaction program
The reaction program is not optimal. If there are non-specific miscellaneous bands smaller than the target band, increase the annealing temperature and reduce the number of cycles. If there are non-specific miscellaneous bands larger than the target band, reduce the extension time and the number of cycles.
Q4: The bands are diffuse or smeared when amplification products are run on a gel.
A4: (1) Gel
Melt the gel completely during preparation.
(2) Primers
Check whether the primers have degraded.
(3) Template
The template is degraded or excessive. Examine the integrity and concentration of the template by electrophoresis, and prepare a new template if necessary. Consult the Instructions for Use for the template input amount. If the target band is long and the cDNA is used as the template, check the purity and integrity of the RNA used for reverse transcription, and perform reverse transcription again without random primers.
(4) Reaction program
The reaction program is not optimal. If the annealing temperature is inappropriate, determine the appropriate annealing temperature by testing a gradient of annealing temperatures. Try the touchdown PCR program.
Q5: There are amplification products in the blank control (NTC) group.
A5: (1) Problematic primer design
The amplified sequence is homologous to the non-target sequence. Thus, non-target sequences are also amplified in PCR.
(2) If the band corresponding to the amplification product has the same size as the target bands, this indicates contamination.
Repeat the experiment with a fresh Mix, water, or primers. Prepare the reaction system on an ultra-clean workbench to minimize aerosol contamination.
(3) To avoid cross-contamination of the target sequence by the genome or large fragments, perform the steps with care to prevent the target sequence from being pipetted into the loading pipette or spilling from centrifuge tubes.
(4) To avoid contamination of the target gene by small airborne nucleic acid fragments, use the nested PCR method to reduce or eliminate contamination.
Q6: Do dyed PCR products affect restriction digestion?
A6: Tests show that dyed PCR products do not affect restriction digestion.
