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PCR reaction setup calculators

PCR, qPCR and LAMP reaction setup calculator; tool for planning PCR, qPCR and LAMP (Loop-mediated Isothermal Amplification) reactions, mixing solutions.

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The higher quality of primers is help to save PCR efficiency at changing PCR conditions. PCR reaction set up in room temperature and performed with or without hot-start enzymes.

The range of optimal annealing temperature (Ta) was calculated Tm of primer or optionally plus 6-12°C, and in practice PCR efficiency was tested with gradient annealing temperature using MasterCycler Gradient (Eppendorf). For primer combinations with very different Tm, the optimal annealing temperature was chosen according to lowest Tm primer (primer with CG content higher then 50% is tolerant to wide annealing Ta, from 55°C up to 72°C).
The optimal annealing temperature for PCR is calculated directly as the value for the primer with the lowest Tm (Tmmin), our empirical formulae:

Ta calculation,
where L is length of PCR fragment.

PCR steps - the primers binding (usually 50-68°C) and the polymerase extension (usually 60-75°C), we recommend to join into one step as 65-72°C. This step includes primer binding the target and polymerase extension at once; the recommended time for this step is 1 second for each 100 bases of PCR product. The denaturation of genomic DNA is easy with short step at 98°C, 5-10 seconds.

The PCR was performed in a 25 µl reaction mixture containing 25 ng DNA, 1x ThermoPol® buffer (with 2 mM MgSO4), 0.2-1 µM of primer (for primer combinations – maximum 2 µM is total concentration), 0.2 mM dNTPs, 0.5 U Taq DNA polymerase and optimal additional 0.005U Pfu DNA Polymerase (for long and efficient PCR products amplification).
A polymerases mix consisting of 100-500 units of Taq (or DyNAzyme™ II, Biotools) DNA polymerase with 1 unit of Pfu DNA Polymerase) greatly increased efficiently of amplification for long bands and the accuracy of the PCR.

ThermoPol® Buffer, 1X: 20 mM Tris-HCl (pH 8.8, 25°C), 2 mM MgSO4, 10 mM KCl, 10 mM (NH4)2SO4, 0.1% Triton® X-100.

The PCR machine was programmed for amplification short and long (2,000-6,000 bases) amplicons:
1 cycle at 95°C 90 sec; 25-30 cycles for 95°C (15 sec), 68-72°C (60-400 sec); and final extension step 72°C 5 min.

Amplification protocol for short (100-2,000 bases) amplicons:
1 cycle at 95°C 90 sec; 25-30 cycles for 95°C (15 sec) and 64-72°C (1-30 sec) and 72°C (1-60 sec); final extension step 72°C 5 min.

Touchdown amplification protocol for short (100-2,000 bases) amplicons:
1 cycle at 95°C 90 sec; 10 cycles for 95°C (15 sec) and 72°C (10-30 sec); 20-25 cycles for 95°C (15 sec) and 60-68°C (10 sec) and 72°C (10-30 sec); final extension step 72°C 5 min.

Loop-mediated Isothermal Amplification (LAMP)

LAMP is an isothermal amplification method designed to detect a target nucleic acid without requiring sophisticated equipment. LAMP provides high sensitivity (to fg or <10 copies of target) but with rapid results: reactions can be performed in as little as 5–10 minutes. Reactions can be performed with limited resources, using a water bath for incubation and detection of results by eye, or with real-time measurement and high-throughput instruments. Detection of RNA targets is accomplished by simple addition of a reverse transcriptase to the LAMP reaction, with RT-LAMP performed as a true one-step, isothermal workflow.

Required Materials:

Optional Materials:

Before You Start:

A LAMP Primer mix can be prepared with all 4 or 6 (with Loop) primers. A 10X Primer Mix should contain: 16 µM FIP, 16 µM BIP, 2 µM F3, 2 µM BE, 4 µM LoopF, 4 µM LoopB in TE or water.

Reactions should be setup on ice. If room temperature setup is desired, use Bst 2.0 WarmStart DNA polymerase.

If analyzing via agarose gel electrophoresis or other method requiring opening LAMP reaction vessels, setup secondary analysis area and equipment to avoid contamination.

Procedure:

Component Volume (25 µl reaction)
10X Isothermal Amplification Buffer 2.5 µL
10 mM dNTPs 3.5 µL (1.4 mM final)
100 mM MgSO4 1.5 µL (6 mM+2 mM in buffer=8 mM final)
10X Primers 2.5 µL (1.6 µM FIP/BIP, 0.2 µM F3/B3, 0.4 µM LoopF/B)
Bst 2.0 (8,000 U/mL) 1 µL (0.32 U/µL)
Sample e.g. 1µL
MilliQ H2O 13 µL
For RNA Targets: WS RTx 0.5 uL

Running a no-template control is strongly recommended to ensure amplification specificity.

If optimization is desired, try titrating concentration of Mg (4–10 mM final) or Bst 2.0 (0.04-0.32 U/µL), or changing reaction temperature (50–72 °C).



The tools are written in Java with NetBeans IDE (Oracle) and require the Java 8 Runtime Environment (JRE) on a computer.

Note: If you don't see the Java Web Start application's Java Web Start button, Java application needs the Java 8 Runtime Environment (JRE) or complete Java Development Kit (JDK), can be downloaded from java.com), and you might need to enable the JavaScript interpreter in your browser so that the Deployment Toolkit script can function properly.

UNDERSTANDING A JAVA ERROR MESSAGE:
If the Java 8 security level is set to "Very High" then no unsigned Java programs are allowed to execute.

Solution:
Add http://primerdigital.com/ site to "Exception Site List", and set "Security Level" to High.
Download a Certificate file, import it to "Signer CA" at Java Control Panel:
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Contact
Dr. Ruslan Kalendar


Reference apply to all Web Tools update, if you use it in your work please cite:

Kalendar R, Khassenov B, Ramankulov Y, Samuilova O, Ivanov KI 2017. FastPCR: an in silico tool for fast primer and probe design and advanced sequence analysis. Genomics. DOI:10.1016/j.ygeno.2017.05.005

Kalendar R, Muterko A, Shamekova M, Zhambakin K 2017. In silico PCR tools a fast primer, probe and advanced searching. Methods in Molecular Biology, 1620: 1-31. DOI:10.1007/978-1-4939-7060-5_1

Kalendar R, Tselykh T, Khassenov B, Ramanculov EM 2017. Introduction on using the FastPCR software and the related Java web tools for PCR, in silico PCR, and oligonucleotide assembly and analysis. Methods in Molecular Biology, 1620: 33-64. DOI:10.1007/978-1-4939-7060-5_2

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