2X PCR Master Mix

Item Number

P743507

• Stability And Storage: Store at -20℃, valid for at least 1 year.
• Storage Temp: Store at -20°C
• Shipped In: Ice chest + Ice pads

Product Description

The 2X PCR Master Mix is a 2X concentrated solution of Taq DNA Polymerase, PCR Buffer, dNTP and all other components required for PCR, except for templates and primers. This premixed formulation simplifies the PCR reaction setup, reduces contamination due to a reduced number of pipetting steps, and thereby improves the reproducibility of PCR reactions.This product can be used for routine PCR amplifications and is especially suitable for qualitative and semi-quantitative PCR amplifications as well as TA cloning of DNA fragments less than 2kb.This product can amplify DNA fragments up to 8 kb, but is more generally used to amplify DNA fragments less than 2kb.This product is highly stable and its activity does not change after 15 repeated freeze-thaws.When a PCR reaction volume of 50μl is used, this product is sufficient for 160 reactions. When a reaction volume of 20µl is used, this product is sufficient for 400 reactions.

Precautions：

Because PCR reaction is extremely sensitive, contamination must be avoided during the preparation of PCR reactions. Negative control without templates is recommended for all PCR assays.Taq DNA polymerase has an error probability of about 2.2×10-5 per cycle in PCR. For cloning DNA fragments larger than 1kb, it is recommended to use DNA polymerase with lower error probability, such as Pfu DNA polymerase and Taq DNA polymerase.Although repeated freeze-thaws up to 15 cycles does not affect the performance of this product, it should still be avoided. Too many cycles of repeated freeze-thaw may decrease the activity of this product.Before using this product, it must be completely thawed and mixed well gently. Avoid the formation of air bubbles.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation.

Instructions for Use：

Set up the PCR reaction:a. Thaw PCR components at room temperature and mix well prior to use. Keep the DNA Polymerase on ice.b. Set up the reaction in a PCR tube on ice as follows:ReagentFinal ConcentrationVolumeVolumeNuclease-free Water-(21-x)μl(8.4-y)μlTemplate DNA10pg-1μg*xμlyμlPrimer Mix (10μM each)0.8μM4μl1.6μl2X PCR Master Mix1X 25μl10μlTotal Volume-50μl20μl* In a 50µl of PCR reaction mix, the template amount required varies for different types of DNA. Use 0.1-1µg of mammalian DNA, 10-100ng genomic DNA from E. coli, or 0.1-10ng of plasmid DNA. Too much DNA template tends to produce nonspecific PCR products. c. Mix well gently by vortex or pipetting. Centrifuge briefly to collect liquid at the bottom of the PCR tube.d. (Optional) When using a thermal cycler without a heated lid, place a drop of mineral oil onto the top of the PCR reaction mixt.2.Transfer PCR reactions to a thermal cycler and run thermocycling conditions as follows: StepTemperatureTimeCyclesInitial denaturation94℃ 3min1Denaturation94℃ 30sec30Annealing55℃ 30secExtension72℃ 1minFinal extension72℃ 10min1Hold4℃ --Note 1: PCR running conditions should be adjusted based on the template, primer sequence, the length of PCR product or GC content, etc.Note 2: The optimal extension time varies depending on the amplicon length. The recommended extension time is 1 min per kb. For example, set the extension time at 1 min for 1kb amplicon, and 2 min for 2 kb amplicon. Note 3: For initial PCR, the number of cycles can be set to 35 to ensure that the expected PCR product can be amplified. The number of cycles for semi-quantitative or quantitative PCR analysis must be optimized appropriately so that the PCR reaction does not reach a plateau.FAQ:1.Few or no specific PCR products.a. It could be due to poor design of primers. Use primer design tools for primer design to avoid inappropriate GC content, secondary structure, dimer, annealing temperature, length, specificity and other possible problems. When adding restriction enzyme cutting sites in the primer sequence, the same problems need to be considered. In the case that positive control primers work normally but not your primers, redesign primers.b. DNA to be amplified may have a high GC content. High GC genes are relatively difficult to be amplified. In such a case, GC-rich buffer suitable for amplifying DNA with high GC content can be used, and PCR reaction parameters should be adjusted accordingly. Direct addition of 1-10% DMSO or 5-20% glycerol is also helpful for amplifying fragments with high GC content.c. Amplification of long fragments. Although Taq DNA polymerase can amplify DNA fragments up to 8 kb, but it is more suitable for amplification of fragments less than 3 kb.d. PCR reactions set up at room temperature tend to produce non-specific bands. It is recommended to set up PCR reactions on ice.e. The presence of secondary structure in primers, primer dimers or short primers, may result in poor annealing of primers to the target sequence. In this case, methods such as touch down can be used to anneal, usually by gradually and slowly lowering the temperature from 65℃ to 55℃ or 50℃ to make the annealing more sufficient. f. The annealing temperature needs to be optimized. If necessary, use a temperature gradient to determine the optimal annealing temperature for each template-primer pair combination. g. Insufficient extension time. The extension time can be extended 2-5 times from the recommended extension time, and can be set to 5 minutes per 1kb for fragments difficult to amplify.h. Insufficient denaturation. To amplify long DNA or high GC DNA fragment, the initial denaturation temperature can be adjusted to 95℃ for 1 min or even 95℃ for 2-4 min.i. Perform PCR reactions on a different thermal cycler to avoid possible problems with the instrument.j. Insufficient number of PCR cycles. Try more PCR cycles, but do not exceed 40 cycles. k. Insufficient amount of template. Add more DNA templates or try nested PCR or secondary PCR. Nested PCR is to design another pair of PCR primers inside the original PCR primers, and then conduct PCR amplification again with the diluted first PCR product as template. Instead, secondary PCR uses the same primers for second PCR amplification with the diluted PCR product as template. Nested PCR usually can remove the non-specific DNA amplification, but secondary PCR cannot.l. DNA sample contains substances that inhibit the PCR reactions. In such a case, template DNA can be purified using appropriate DNA purification methods such as column purification.m. When non-specific fragments are produced, the annealing temperature can be increased appropriately.n. Positive and negative controls are always recommended when optimizing PCR reactions.Related Products:产品编号产品名称包装D7205Taq DNA Polymerase200UD7207Taq DNA Polymerase1000UD7209Taq DNA Polymerase5000UD7216Pfu DNA Polymerase100UD7217Pfu DNA Polymerase1000UD7218Taq DNA Polymerase100UD7219Taq DNA Polymerase1000UD7220 DNA Polymerase200UD7221 DNA Polymerase1000UD7222 Plus DNA Polymerase200UD7222B Plus DNA Polymerase1000UD72282X PCR Master Mix400TD7232PCR Kit with Taq400TD7233PCR Kit with Taq2000TD7237PCR Kit with Taq200T