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Products for PCR, FAQ
Q 01: Do DyNAzyme™ I, II and EXT DNA Polymerases, and Phusion™ Polymerase add the non-template dependent 3'-A overhang?
DyNAzyme™ DNA Polymerases are capable of adding a 3'-overhang. However, the efficiency at which the extra base is added is sequence-dependent. The extra base is usually A, however, the enzyme can also incorporate other bases or even more than a single base at a sequence-spesific manner. To enhance TA-cloning it may be helpful to include an additional incubation period of 10-30 minutes at 72 °C to the end of cycle protocol.
Phusion™ High-Fidelity DNA Polymerase generates blunt end products therefore blunt end cloning is recommended. If TA cloning is required, it can be performed by adding A overhangs to the blunt PCR product with e.g. DyNAzyme™ II DNA Polymerase (F-501S/L). However, before adding the overhangs it is very important to remove all the Phusion DNA Polymerase by purifying the PCR product carefully, as the proofreading activity in Phusion DNA Polymerase is very strong. Any remaining Phusion DNA Polymerase will degrade the A overhangs, thus creating blunt ends again. For a detailed protocol see Finnzymes' DNA Polymerases and cloning.
Q 02: What nucleotide analogues can I use with DyNAzyme™ and Phusion™ DNA Polymerases?
DyNAzyme™ I and II DNA Polymerases can use dUTP, biotinylated dNTPs, 7-deaza-dGTP, digoxigenin-dUTP, bromo-dUTP, radiolabeled dNTPs and ITP. DyNAzyme™ EXT DNA Polymerase and Phusion ™ DNA Polymerase can't read dUTP-derivatives or dITP in the template strand so the use of these analogues is not recommended.
Q 03: How should I design a set of PCR-primers
1. Try to avoid complementarity between the primers to prevent primer-dimer formation.
2. Try to avoid inverted repeats (self-complementarity).
3. CG-content of the primer should be around 50%.
4. Try to avoid G's and C's at the 3'-end of the primers.
There are many computer programs which will help you to design a primer-pair. For calculating the Tm values we recommend to use Finnzymes' Tm calculator.
Q 04: How do I determine the appropriate annealing temperature?
The most computer programs used to design primers will give you the optimal annealing temperature. One easy way is to follow this link: Tech. support links Tm determination
Q 05: How can I facilitate the amplification of templates with hairpin-loop structures or high CG-content?
You can try 5-10 % DMSO, up tp 10 % glycerol, 1-2 % formamide or combinations of these. Note: The use of co-solvents will lower the optimal annealing temperatures of your primers (e.g. 5.5-6 °C in 10 % DMSO). You could also try 7-deaza-dGTP in conjuction with normal dGTP in order to destabilize difficult structures. Note: 7-deaza-dGTP attenuates the signal of ethidium bromide staining.
Q 06: Does DMSO inhibit DyNAzyme™ DNA Polymerases?
10 % DMSO will inhibit DyNAzyme DNA Polymerases to a some extent but you can overcome the inhibition using twice the amount of DyNAzyme DNA Polymerase.
Q 07: Can I do PCR directly from bacterial colonies?
Yes, simply scrape a tiny amount of a single colony and transfer to an empty PCR-tube. Add PCR-mix (20-50 µl) and start cycling using 3 minutes at 94 °C initial denaturation. Triton® X-100 present in reaction buffer will facilitate the lysis of bacterial cells.
Q 08: What is Hot Start PCR?
Phusion™ Hot Start DNA Polymerase and DyNAzyme™ II Hot Start DNA Polymerase are inactive at room temperature, which improves the specificity of PCR by preventing the extension of non-specifically bound primers. The activity of DyNAzyme™ II Hot Start DNA Polymerase is recovered by a 10-minute incubation step at 94°C. Phusion™ Hot Start DNA Polymerase is inactivated by a reversibly bound, specific Affibody® protein, which releases the polymerase fully active at polymeration temperature. Thus, Phusion™ Hot Start DNA Polymerase does not require any additional activation step prior to PCR. The use of Phusion Hot Start DNA Polymerase and DyNAzyme II Hot Start DNA Polymerase is also beneficial when using primers that are prone to primer-dimer formation or when using robotic PCR systems that include incubations at room temperature prior to temperature cycling.
More about Phusion™ Hot Start DNA Polymerase.
More about DyNAzyme™ II Hot Start DNA Polymerase.
Alternatively you can use DyNAwax™ Wax. The wax separates reaction components in reaction prior to heating.
More about DyNAwax™ Wax.
Q 09: How important is the quality of my DNA template in long PCR?
Template preparation becomes particularly important when performing longer amplifications (>15 kb). Therefore, it is recommended to check the lenght of the DNA by agarose gel electrophoresis.
Q 10: What kind of reaction tubes are recommended?
We recommend thin-walled tubes especially for long PCR.
Q 11: What is two-step PCR?
If the Tms of your primers are high enough (over 65°C), the annealing and the extension steps can be combined into a single step.
Q 12: What causes the smearing of the whole lane upon gel electrophoresis?
When long primer extension times are used, the result might sometimes be a smear. The critical factors in these cases are magnesium concentration, enzyme concentration and dNTP concentration. All of the above factors affect each other, so it is possible to achieve the same result with different factor combinations. To avoid smearing, we recommend:
- decrease magnesium concentration (e.g. 0.2 mM steps)
- use less enzyme
- increase dNTP concentration
- shorten extension times
- reduce cycle numbers
Q 13: Does DyNAzyme™ II DNA Polymerase differ from DyNAzyme™ I DNA Polymerase?
Basically the only difference is the source of the enzyme: Thermus brockianus for DyNAzyme I DNA Polymerase and recombinant Escherichia coli strain for DyNAzyme II DNA Polymerase.
Q 14: I want to dilute my enzyme, which buffer should I use?
If you want to store the diluted enzyme, storage buffer should be used (see the data sheet for composition). Otherwise either 1x reaction buffer or even sterile water can be used. If storage buffer is not used, prepare the dilution just before use and do not store it.
Q 15: I need a very high fidelity, what reaction conditions should I use?
Best fidelities are obtained when Finnzymes' unique proofreading polymerase, Phusion™ High-Fidelity DNA Polymerase, is used in HF buffer.
Q 16: Do you have any publications about DyNAzyme™ DNA Polymerases?
Yes, see the reference list.
Q 17: Taq is working but DyNAzyme™ DNA Polymerase is not, why?
Sometimes DyNAzyme DNA Polymerase is working and Taq is not. This simply reflects that the enzymes are different and often the optimal reaction conditions are not the same for both enzymes. By optimizing the reaction conditions this situation can usually be solved (Mg-concentration, amount of enzyme, dNTP concentration, primer concentration, annealing temperature, co-solvents, etc.).
Q 18: My negative control is giving a smear or a band upon electrophoresis. Is DyNAzyme™ DNA Polymerase contaminated with DNA?
DNA polymerases bind DNA very tightly. All polymerases contain minute amounts of DNA. DyNAzyme DNA Polymerase is very extensively purified and the amount of DNA is as low as it can get. One possibility is that the enzyme may have got contaminated upon opening in your laboratory.
Q 19: What is touchdown PCR?
It is a method for increasing specificity of PCR reactions. Touchdown PCR uses a cycling program where the annealing temperature is gradually reduced e.g. 1-2 °C/every second cycle. The initial annealing temperature should be several degrees above the estimated Tm of the primers. Annealing temperature is then gradually decreased until it reaches the calculated annealing temperature of the primers or some degrees below. Amplification is then continued using this annealing temperature.
Q 20: Does Phusion™ Hot Start DNA Polymerase need a separate activation step in the PCR protocol?
No. Phusion™ Hot Start DNA Polymerase is inactivated by a reversibly bound, specific Affibody® protein, which releases the polymerase fully active at polymerization temperature.
Q 21: Can protocols optimized for Phusion™ DNA Polymerase directly be applied to Phusion™ Hot Start DNA Polymerase?
In many cases yes. However, with Phusion™ Hot Start DNA Polymerase, use primers with Tm 60 ºC or higher. Typically the length of such primers is 20 nt or more. Calculate the Tm values with the nearest-neighbor method (see a link to a calculator below). In addition, use of detergent free reaction buffers is not recommended with Phusion™ Hot Start DNA Polymerase.
Tm calculator for PCR.
