PCR
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Cycling Conditions | Cycling Conditions | ||
| - | Our PCR product will be long 882bp and in this case the Tm of primers Fw is | + | Our PCR product will be long 882bp and in this case the Tm of primers Fw is 69.5°C and Rev is 68.1°C so we decided to adopted as annealing temperature :59°C |
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Latest revision as of 09:02, 19 October 2007
Protocol for PCR with Taq DNA Polymerase Avoiding Contamination PCR allows the production of more than 10 million copies of a target DNA sequence from only a few molecules. The sensitivity of this technique means that the sample should not be contaminated with any other DNA or previously amplified products (amplicons) that may reside in the laboratory environment.
DNA sample preparation, reaction mixture assemblage and the PCR process, in addition to the subsequent reaction product analysis, should be performed in separate areas. A Laminar Flow Cabinet equipped with a UV lamp is recommended for preparing the reaction mixture. Fresh gloves should be worn for DNA purification and each reaction set-up. The use of dedicated vessels and positive displacement pipettes or tips with aerosol filters for both DNA sample and reaction mixture preparation, is strongly recommended. The reagents for PCR should be prepared separately and used solely for this purpose. Autoclaving of all solutions, except dNTPs, primers and Taq DNA Polymerase is recommended. Solutions should be aliquoted in small portions and stored in designated PCR areas. Aliquots should be stored separately from other DNA samples. A control reaction, omiting template DNA, should always be performed, to confirm the absence of contamination. These are only rough guidelines. Detailed instructions about PCR laboratory setup and maintenance may be found in PCR Methods and Applications, 3, 2, S1-S14, 1993. | Preparation of Reaction Mixture
To perform several parallel reactions, prepare a master mix containing water, buffer, dNTPs, primers and Taq DNA Polymerase in a single tube, which can then be aliquoted into individual tubes. MgCl2 and template DNA solutions are then added. This method of setting reactions minimizes the possibility of pipetting errors and saves time by reducing the number of reagent transfers.
Reaction Mixture Set Up
Gently vortex and briefly centrifuge all solutions after thawing.
Add, in a thin-walled PCR tube, on ice:
| Reagent ; | Final concentration | Quantity, for 50 µlof reaction mixture |
|---|---|---|
| Sterile deionized water | - | variable |
| 10X Taq buffer | 1X | 5 µl |
| 2 mM dNTP mix | 0.1-1 µM | variable |
| Primer I | 0.1-1 µM | variable |
| Primer II | 1.25 u / 50 µl | variable |
| Taq DNA Polymerase | 1.25 u / 50 µl | variable |
| 25 mM MgCl2 | 1-4 mM | variable* |
| Template DNA | 10pg-1 µg | variable |
* Table for selection of 25 mM MgCl2 solution volume
| Final concentration of MgCl2 in 50 µl reaction mix, mM | 1.0 | 1.25 | 1.5 | 1.75 | 2.0 | 2.5 | 3.0 | 4.0 |
|---|---|---|---|---|---|---|---|---|
| Volume of 25 mM MgCl2, µl | 2 | 2.5 | 3 | 3.5 | 4 | 5 | 6 | 8 |
Gently vortex the sample and briefly centrifuge to collect all drops from walls of tube. Overlay the sample with half volume of mineral oil or add an appropriate amount of wax. This step may be omitted if the thermal cycler is equipped with a heated lid. Place samples in a thermocycler and start PCR. Components of the Reaction Mixture
PCR FOX3 PROMOTER FROM GENOMIC DNA
Usually the amount of template DNA is in the range of 0.01-1 ng for plasmid or phage DNA and 0.1-1 µg for genomic DNA, for a total reaction mixture of 50 µl. Higher amounts of template DNA usually increase the yield of nonspecific PCR products, but if the fidelity of synthesis is crucial, maximal allowable template DNA quantities together with a limited number of PCR cycles should be used to increase the percentage of "correct" PCR products.
Finally volume is 100μl
| Acqua, µl | 140 |
|---|---|
| Buffer, µl | 20 |
| MgCl2, µl | 6 |
| dNTPs, µl | 4 |
| Taq, µl | 2 |
| PrimerFw, µl | 10 |
| PrimerRev, µl | 10 |
| Template, µl | 8 |
Cycling Conditions
Amplification parameters depend greatly on the template, primers and amplification apparatus used. Our PCR product will be long 832pb and we calculated the approx. melting temperature (Tm) using the following formula:
Tm= 4 (G + C) + 2 (A + T)
G, C, A, T - number of respective nucleotides in the primer. Annealing temperature should be approx. 5°C lower than the melting temperature. In our case the Tm of primers Fw is 68°C and Rev is 63°C so we decided to adopted as annealing temperature : 56°C
| I°CYCLE | 94°c for 5 minutes | ||
|---|---|---|---|
| 25 CYCLES | denaturation 94°C for 30 seconds | annealing 56°c for 1 minutes | elongation 72°c for 1 minutes |
| LAST CYCLE | 72°C for 2 seconds | 4°c |
PCR FIREFLY LUCIFERASE FROM PLASMID p2UG-2XORE-LUC Finally volume is 100μl
| Acqua, µl | 140 |
|---|---|
| Buffer, µl | 20 |
| MgCl2, µl | 6 |
| dNTPs, µl | 4 |
| Taq, µl | 2 |
| PrimerFw, µl | 10 |
| PrimerRev, µl | 10 |
| Template, µl | 8 |
Cycling Conditions
Our PCR product will be long 1653pb and in this case the Tm of primers Fw is 68,2°C and Rev is 69,5°C so we decided to adopted as annealing temperature : 58°C
| I°CYCLE | 94°c for 5 minutes | ||
|---|---|---|---|
| 25 CYCLES | denaturation 94°C for 30 seconds | annealing 58°c for 1 minutes | elongation 72°c for 1 minutes |
| LAST CYCLE | 72°C for 2 seconds | 4°c |
PCR 2XORE-CYC Tata FROM PLASMID p2UG-2XORE-LUC Finally volume is 100μl
| Acqua, µl | 140 |
|---|---|
| Buffer, µl | 20 |
| MgCl2, µl | 6 |
| dNTPs, µl | 4 |
| Taq, µl | 2 |
| PrimerFw, µl | 10 |
| PrimerRev, µl | 10 |
| Template, µl | 8 |
Cycling Conditions
Our PCR product will be long 292pb and in this case the Tm of primers Fw is 58,4°C and Rev is 60°C so we decided to adopted as annealing temperature :54°C
| I°CYCLE | 94°c for 5 minutes | ||
|---|---|---|---|
| 25 CYCLES | denaturation 94°C for 30 seconds | annealing 54°c for 45 seconds | elongation 72°c for 1 minutes |
| LAST CYCLE | 72°C for 2 minutes | 4°c |
PCR 1XORE-CYC Tata FROM PLASMID p2UG-2XORE-LUC Finally volume is 100μl
| Acqua, µl | 65 |
|---|---|
| Buffer with MgCl2, µl | 20 |
| dNTPs, µl | 2 |
| phusion, µl | 1 |
| PrimerFw, µl | 5 |
| PrimerRev, µl | 5 |
| Template, µl | 2 |
Cycling Conditions
Our PCR product will be long 292pb and in this case the Tm of primers Fw is 58,4°C and Rev is 60°C so we decided to adopted as annealing temperature :54°C
| I°CYCLE | 98°c for 30 seconds | ||
|---|---|---|---|
| 30 CYCLES | denaturation 98°C for 10 seconds | annealing 58°c for 30 seconds | elongation 72°c for 30 seconds |
| LAST CYCLE | 72°C for 10 minutes | 4°c |
PCR pho 80 from DNA of yeast
Finally volume is 200μl
| Acqua, µl | 140 |
|---|---|
| Buffer, µl | 20 |
| MgCl2, µl | 6 |
| dNTPs, µl | 4 |
| Taq, µl | 2 |
| PrimerFw, µl | 10 |
| PrimerRev, µl | 10 |
| Template, µl | 8 |
Cycling Conditions
Our PCR product will be long 882bp and in this case the Tm of primers Fw is 69.5°C and Rev is 68.1°C so we decided to adopted as annealing temperature :59°C
| I°CYCLE | 94°c for 5 minutes | ||
|---|---|---|---|
| 25 CYCLES | denaturation 94°C for 30 seconds | annealing 59°c for 1 minutes | elongation 72°c for 1 minutes nd 30 seconds |
| LAST CYCLE | 72°C for 2 minutes | 4°c |
