2×LA PCR Master Mix (for PCR series)

Product Information

Product Description/Introduction

This product is a 2× premix solution containing LA DNA polymerase, reaction buffer and dNTPs, which facilitates the rapid preparation of PCR reaction system. LA DNA Polymerase is a hybrid enzyme consisting of a modified Taq DNA Polymerase and a DNA Polymerase containing a 3'5' exonuclease activity. It is capable for robust fidelity up to 6 times that of Taq DNA polymerase and extension speed of approximately 10 s/kb. The combination of rapid amplification and high continuous synthesis capability with specially optimized reaction buffer can amplify target DNA fragments up to 30 KB from the genome, and have high amplification efficiency for different source and different length templates. For target fragments less than 10kb, the yield is significantly increased. The PCR products are blunt end with A base, convenient for T-Vector cloning. This product contains two tracer dyes and can be directly load on gel for agarose gel electrophoresis.

Storage and Shipping Conditions

Ship with wet ice; Store at -20, valid for 12 months.

Product Contents

Assay Protocol / Procedures

1. Recommended PCR reaction system

a: If the template is plasmid or from bacteriophage, 10 ng-1 pg added in 50 μL rnx is recommended. If tempalted is genomic DNA, 250ng-50ng added in 50 μL rnx is recommended. If template is cDNA, dilute your original cDNA by 2-100 times, and the volume of diluted cDNA added to reaction is no more than 10% of total reaction volume. Higher amounts of template tend to lead to non-specific amplification. Lower amounts of template tend to lead to low PCR amplification efficiency.

b: The range of final primer concentration used is 0.2-1.0 μM，and 0.4 μM is recommended. Too little primer may result in low or no amplification yield, and too much primer may result in non-specific amplification.

c: For high GC content template, DMSO with no more than 10% total volume can be added.

2. Recommended PCR amplification conditions

a: The pre-denaturation time of 30 s is suitable for most conventional templates, and the denaturation time of complex templates can be extended to 2 min;

b: The short denaturation time is favorable for the amplification of long fragments over 20 kb;

c: For complex templates, the annealing time can be extended to 30 s for amplification with annexed primers. When the annealing temperature is between 68 and 70, the amplification specificity can be significantly improved by combining the annealing/extension temperature into annealing temperature;

d: Simple templates such as plasmids recommend an extension speed of 5-10s/kb; The recommended elongation rate for conventional genomic templates is 10-15s/kb, and complex template 15-30s/kb.

Note

1. For different templates, the amount of template needs to be appropriate and adjusted as required.

2. The number of dNTPs and primers can be adjusted for amplification of fragments over 15 KB, and the designed length of primers is 25~35 bp. It is recommended to use kit to extract high-quality templates.

3. Different PCR apparatus and PCR reaction tube will also affect the amplification of long fragment.

4. Primer design needs to follow primer design principles.

5. For complex templates, it is necessary to adjust the annealing temperature and extend the extension time to achieve efficient amplification.

5. Thoroughly thaw and mix before using. After complete melting, it can be stably stored at 4 for at least 2 weeks, avoiding freeze-thaw cycles.

6. For your safty and health,please wear safety glasses, gloves, or protective clothing.

For Research Use Only!

https://www.servicebio.com/goodsdetail?id=6756