Which DNA polymerase do Z-PCR Mix, 5x Z-PCR Mix, Direct-Load PCR Mix and 5xDirect-Load PCR Mix contain?

Z-PCR Mix, 5xZ-PCR Mix, Direct-Load PCR Mix and 5x Direct-Load PCR Mix contain TaqDNA polymerase.

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What is Z-PCR Mix?

Z-PCR Mix is a complete ready-to-use reaction mixture optimized for efficient amplification of DNAs (genomic, plasmid, phage, or viral DNA) by PCR. Users only need to add template and primers to set up a PCR reaction. Z-PCR Mix is designed to save time for setting up PCR reactions and reduce the risk of contamination.

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What is 5xZ-PCR Mix?

5xZ-PCR Mix is a 5x ready-to-use reaction mixture optimized for efficient amplification of DNA (genomic, plasmid, phage, or viral DNA) by PCR. Users only need to add a template, water and primers to set up a PCR reaction. 5xZ-PCR Mix contains stabilizers and loading buffer without dye, so that PCR products can be loaded directly onto an agarose gel after PCR. It is designed to save time when setting up PCR reactions and reduce the risk of contamination. The 5xZ-PCR Mix has the following advantages: a very diluted template DNA can be used in a PCR amplification and 5xZ-PCR Mix is more stable than 2x and 1.1x PCR Master Mixes.

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What is Direct-Load PCR Mix?

Direct-Load PCR Mix contains all the components including loading dye (bromophenol blue) for PCR with the exception of a template and primers.   The advantage of Direct-Load PCR Mix is that samples can be loaded directly onto an agarose gel after PCR.

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What is 5xDirect-Load PCR Mix?

5xDirect-Load PCR Mix is a newly formulated PCR Master Mix that improves the stability of enzymes and other components. The 5x Direct-Load PCR Master Mix is an optimized ready-to-use 5x mixture containing everything (except a DNA template and primer set) needed for efficient amplification of a template DNA (up to 3.5 kb) in PCR. The 5x Direct-Load PCR Master Mix is designed to save time when setting up reactions, improve stability and reduce the risk of contamination. The 5x Direct-Load PCR Master Mix contains stabilizers and loading dye (bromophenol blue) so that PCR products can be directly loaded onto agarose or polyacrylamide gels. PCR products can also be digested with enzymes or used for ligation without prior removal of the loading dye. The 5x Direct-Load PCR Master Mix is more stable than 2x and 1.1x PCR Master Mixes.

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What is Z-PCR Mix GC-Rich?

Z-PCR Mix GC-Rich is  a complete ready-to-use reaction mixture specifically optimized for efficient amplification of GC-rich and/or problematic templates by PCR. Z-PCR Mix GC-Rich contains enhancing agents and stabilizers that can lower the Tm of the template.   Z-PCR Mix GC-Rich has been successfully tested for specific amplification of a 72.7% GC-rich region in human genomic DNA. Z-PCR Mix GC-Rich is designed to save time when troubleshooting, improve stability and reduce the risk of contamination.

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How does Z-PCR Mix GC-Rich compare with PCR Master Mixes from other companies for the amplification of GC-rich templates?

Z-PCR Mix GC-Rich has been compared with PCR Master Mixes from other companies for amplification of a 72.7% GC-rich region (348bp) in human genomic DNA.   Results show that three out of four PCR Master Mixes from other companies did not yield a specific DNA band (Figure 1, Samples 3, 4, and 6).   Z-PCR Mix GC-Rich (Sample 2) gives a much higher yield than the PCR Master Mix from Company A (Sample 5) under the same conditions. 
        Z-BioMed  Other Companies Figure 1.  Comparison of different PCR Mixes for
          M      1     2      3     4      5     6     (-)  the amplification of a 72.7% GC-rich region (348bp) in human genomic DNA.
1  Direct-Load PCR Mix GC-Rich       Z-BioMed
2  Z-PCR Mix GC-Rich                        Z-BioMed
3  PCR Master Mix                             Company G
4  PCR Master Mix                             Company P
5  PCR Master Mix                             Company A
6  PCR Master Mix                             Company B

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What advantages do your Direct-Load Colony PCR Screening Kits have?

The Direct-Load Colony PCR Screening Kits are designed for rapid screening of colonies using PCR. Each kit contains everything needed for colony PCR screening and 1 of the 8 primer sets compatible with the most commonly used vectors. These kits can be used with bacterial colonies, bacterial cultures, and plasmid DNAs. The colony PCR products can be directly loaded onto agarose gel to check inserts. It takes about 3 hours using these kits to screen colonies for inserts compared to 1 to 2 days using the traditional method of DNA minipreps and restriction enzyme digestions. The cost of a Direct-Load Colony PCR Screening Kit is less than the cost of a Plasmid MiniKit, and a Direct-Load Colony PCR Screening Kit can be used for 100 samples, compared to 25 for a Plasmid MiniKit.

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How many copies of the template should I use for my PCR?

Successful amplification of the specific target is dependent on the amount and quality of the template DNA. The amount of template needed for successful PCR is dependent on the complexity of the sample. Start with 102 to 105 copies of the template for your PCR if possible.

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How do I calculate the number of molecules needed for a successful PCR for my sample?

You can use the following table (Table 1) to figure out how many molecules per µg or ng of your samples, and then calculate the amount of samples required for a successful PCR (102 to 105 molecules).
Table 1.  Molecules Per Microgram (µg) or Nanogram (ng) of Samples

Source Base pairs/ haploid genome Molecules per microgram (µg) Molecules per nanogram (ng)
1 kb dsDNA _ 9.12x 1011 9.12x 108
1 kb ssDNA _ 1.82x 1012 1.82x 109
1 kb RNA _ 1.77x 1012 1.77x 109
Lambda DNA 48,502 1.90x 1010 1.90x 107
E.coli  genomic DNA 4.7x 106 1.94x 108 1.94x 105
S. cerevisiae genomic DNA 1.21x 107 7.54x 107 7.54x 104
C. elegans genomic DNA 9.7x 107 9.40x 106 9.40x 103
A. thaliana 1.0x 108 9.12x 106 9.12x 103
Drosophila genomic DNA 1.8x 108 5.07x 106 5.07x 103
Mouse genomic DNA 3.0x 109 3.04x 105 3.04x 102
Rat genomic DNA 3.0x 109 3.04x 105 3.04x 102
Human genomic DNA 3.0x 109 3.04x 105 3.04x 102

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What concentration of primers should I use for my PCR?

0.1 µM to 1.5 µM of each primer should be used for a PCR.

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How to convert pmoles of primers to µM of primers?
1 pmole/µl = 1 µM; 1 nmole/µl = 1 mM

For example, if you have 60500 pmoles (60.5 nmoles) of primer, you can add 60.5 µl of dH20 to make  a 1mM stock solution of primer (60.5 nmoles + 60.5 µl of dH20 = 1 mM solution of primer).
Dilute 1:100 = 10 µM
If you use 1 µl of 10 µM primer in 25 µl PCR reaction, your final primer concentration will be 0.5 µM.

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When should I use a Taq DNA polymerase?

If you want to amplify DNA fragments efficiently, you should use a Taq DNA polymerase.  Z-PCR Mix, Direct-Load PCR Mix and Z-PCR Mix GC-Rich  contain Taq  DNA polymerase at the optimized concentration which can give a high efficiency and yield.  Nonproofreading enzymes such as Taq  DNA polymerase can give you minimum errors and maximum efficiency under appropriate high-fidelity conditions.  High-fidelity conditions include low magnesium and dNTP concentrations (<50µM), and short extension times.  It is very hard to beat Taq DNA polymerase for the combination of specificity, sensitivity, and yield.

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When should I use proofreading enzymes such as Pfu, Pwo, Vent or Deep Vent?

If you need a short fragment from a plasmid, use a high template ( i.e ., ng of plasmid) and keep the cycle number low (10 or 15), then it is fine to use proofreading DNA polymerases.  However, you can not assume that the PCR product will have 100% fidelity just by using a proofreading DNA polymerase.  No DNA polymerase is perfect.  That is, when fidelity is important you still have to sequence your clone no matter how accurate the proofreading DNA polymerase is.

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How does Taq DNA polymerase compare with proofreading enzymes such as Pfu, Vent and Deep Vent?

The properties of Taq and other proofreading DNA polymerases are listed in Table 2.
Table 2. Comparison of Taq and Some Proofreading DNA polymerases

DNA Polymerase Taq Pfu Vent Deep Vent
Organism Thermus Aquaticus Pyrococcus Furiosus Thermococcus Litoralis Pyrococcus Species GB-1
5'-3' Exonuclease Activity Yes No No No
3'-5' Exonuclease Activity No Yes Yes Yes
PCR DNA Ends 3'-A Blunt 95% Blunt 95% Blunt
Yield + + + + + + + + + +
Error Rate (error/bp incorporated) 8 x 10-6 1.3 x 10-6 2.8 x 10-6 2.7 x 10-6

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Why are yields always lower with high-fidelity DNA polymerases (proofreading enzymes)?

Because these high-fidelity DNA polymerases have the 3’ to 5’ exonuclease proofreading activity to degrade single-stranded DNA.  The primers are single-stranded DNA which means that the specificity designed into primer can be changed during the course of the PCR.   However,  that is not the only time when high-fidelity DNA polymerase (proofreading activity) contributes negatively in PCR.   At the end of each cycle, after the DNA polymerase has finished extension and you have a completed duplex, the proofreading activity can remove the last few incorporated nucleotides and  then because there is dNTP present, the polymerase activity repair the nucleotide back in. The problem comes at the end of extension when you heat to denature the duplex you have generated. You denature, and you now have a hyperthermophilic polymerase with an inherent proofreading activity and the ends of your PCR product have been denatured.   Until it is cooled down and primer has bound, that is during that temperature transition of up to 95ºC and  back to the annealing temperature,  50-65ºC,  the proofreading DNA polymerase can be chewing at the 3’ end of the primer extension product which it last synthesized.  PCR products can come up and then disappear.  The issue is important when you have something that is very low-copy number and you have to do 30 to 40 cycles to get a cloneable product level because of very low copy number; it becomes a challenge.

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How can I calculate the percentage of PCR-induced mutants in my PCR products?

The percentage of PCR-induced mutants in final PCR products is mainly dependent on the type of DNA polymerase, PCR target size and cycling number. You can use the following PCR Mutation Calculator to calculate the percentage of PCR-induced mutants in your PCR products.

PCR Mutation Calculator

To calculate the percentage of PCR-induced mutants with different polymerases such as Taq, Pfu, Vent and Deep Vent in any PCR products, enter the target size and cycling number to the following table.

  Enter PCR target size (e.g. 200bp): 
  Enter cycling number (e.g.  30): 
         





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