PCR

From 2007.igem.org

(Difference between revisions)
Line 98: Line 98:
Amplification parameters depend greatly on the template, primers and amplification apparatus used.
Amplification parameters depend greatly on the template, primers and amplification apparatus used.
-
Our PCR product will be long 830pb and we calculated the approx. melting temperature (Tm) using the following formula:
+
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)
                                             Tm= 4 (G + C) + 2 (A + T)
Line 104: Line 104:
G, C, A, T - number of respective nucleotides in the primer.
G, C, A, T - number of respective nucleotides in the primer.
Annealing temperature should be approx. 5°C lower than the melting temperature.
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 : 58°C
+
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
{| border="1"
{| border="1"
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! 25 CYCLES  
! 25 CYCLES  
| denaturation  94°C for 30 seconds
| denaturation  94°C for 30 seconds
-
| annealing    58°c for 1 minutes
+
| annealing    56°c for 1 minutes
| elongation    72°c for 1 minutes
| elongation    72°c for 1 minutes
|-
|-
Line 152: Line 152:
Cycling Conditions
Cycling Conditions
-
Our PCR product will be long 1653pb and in this case the Tm of primers Fw is °C and Rev is °C so we decided to adopted as annealing temperature : °C
+
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
{| border="1"
{| border="1"
Line 169: Line 169:
|}
|}
-
''PCR 1ORE-CYC Tata FROM PLASMID p2UG-2XORE-LUC''
+
''PCR 2XORE-CYC Tata FROM PLASMID p2UG-2XORE-LUC''
Finally volume is 100μl
Finally volume is 100μl
 +
{| border="1"
 +
|-
 +
! 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 °C and Rev is °C so we decided to adopted as annealing temperature : °C
 +
 +
{| border="1"
 +
|-
 +
! I°CYCLE
 +
| 94°c for 5 minutes
 +
|-
 +
! 25 CYCLES
 +
| denaturation  94°C for 30 seconds
 +
| annealing    °c for 1 minutes
 +
| elongation    72°c for 1 minutes
 +
|-
 +
! LAST CYCLE
 +
| 72°C for 2 seconds
 +
| 4°c
 +
|}

Revision as of 14:11, 20 September 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 °C and Rev is °C so we decided to adopted as annealing temperature : °C

I°CYCLE 94°c for 5 minutes
25 CYCLES denaturation 94°C for 30 seconds annealing °c for 1 minutes elongation 72°c for 1 minutes
LAST CYCLE 72°C for 2 seconds 4°c
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