PCR- Optimization of Annealing Temperature

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PCR- Optimization of Annealing Temperature

6 Component Final concentration Taq polymerase 0 5–2 0 units, ideally 1 25 units Deoxy-nucleotides (dNTPs) Typical concentration is 200 µM of each dNTP Magnesium Concentration 1 5-2 0 mM is optimal for Taq DNA Polymerase *

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BCH361-Practical

PCR-Optimization of Annealing

Temperature

2 3

Identification

the location of the target sequence in the

DNA template

Primer design

and primer specificity PCR optimization

Post-PCR

analysis results using agarose gel electrophoresis (AGE) PCR troubleshooting

Start your PCR

and visualize the results by AGE

Next lab

PCR optimization ?

What if not?

REMEMBER:

ÎThere is no single set of conditions that is optimal for all PCR reactions. WHY? 5

PCR components

concentration

Thermal cycling

condition

Duration of the

steps

Temperatures

Number of

cycles 6

ComponentFinal concentration

Taq polymerase0.52.0 units, ideally 1.25 units.

Deoxy-nucleotides(dNTPs)Typical concentration is 200 µM of each dNTP. Magnesium Concentration1.5-2.0 mMis optimal for TaqDNA Polymerase.*

Forward PrimersTypically 0.1-0.5 µM .

Reverse PrimerTypically 0.1-0.5 µM.

DNA Template1ng1µg of genomic templates.

7 It is important to note that while optimization of one parameter, other parameters should be fixed and not changed. WHY ? ÎHow you will know that you reached to the optimum conditions? 8 9

StepTemperatureDuration Cycle

Initial denaturation9497 C3 minx1

Denaturation 9497 C30 sec

x (25-35)Annealing 50
65

C30 sec

Elongation 72-80 C30-60 sec

Final elongation 75-80 C5-7 minx1

Stage 1

Stage 2

Stage 3

10 Reaching the optimum Ta is critical for reaction specificity, as non-specific products may be formed as a result of non-optimal Ta. HOW?

ÎOptimization done by applying

temperature gradient PCR, where PCR carried with different Ta starting at 5 C below the lowest calculated melting temperature (Tm) of the primer pair.

Example.

11 When optimizing Ta what you should do with other PCR component? ÎStart by applying the standard concentration of PCR component that work with majority of

PCR reaction.

12 13

Optimization of PCR annealing temperature.

Be familiar with PCR technique and thermal cycler device. 14 15

1. Start by applying the standard concentration of PCR component that work with majority of

PCR reaction. Use the table to calculate the needed volume of each PCR component: C 1 x V 1 = C 2 x V 2 16

2. Prepare a master mix that contains everything except the DNA template by multiplying the

volume per reaction of each component by (number of desired reaction +1 for pipetting error): 17

3. Using special PCR tubes, distribute the master mix by pipetting ---µl to eahtube.

4. Add the DNA template for each template.

5. Centrifuge the tubes briefly.

6. Set the thermal cycling condition as following:

3. Try different 8 annealing temperatures depending on your primer pair Tm.

4. Set the final volume in the thermal cycler to be 50 µl.

5. Start PCR !!

Initial denaturation

All the rows have the

same temperature (94 -97 C)

Denaturation

All the rows have the

same temperature (94 -97 C)

Annealing

Each raw/column

will have different annealing

Extension

All the rows have the

same temperature (72 -80 C) OR 20 Analyse the results using 2% agarose gel, and determine the optimum Ta.quotesdbs_dbs12.pdfusesText_18

[PDF] PCR- Optimization of Annealing Temperature

BCH361-Practical

PCR-Optimization of Annealing

Temperature

Identification

DNA template

Primer design

Post-PCR

Start your PCR

Next lab

PCR optimization ?

What if not?

REMEMBER:

PCR components

Thermal cycling

Duration of the

Temperatures

Number of

ComponentFinal concentration

Taq polymerase0.52.0 units, ideally 1.25 units.

Forward PrimersTypically 0.1-0.5 µM .

Reverse PrimerTypically 0.1-0.5 µM.

DNA Template1ng1µg of genomic templates.

StepTemperatureDuration Cycle

Initial denaturation9497 C3 minx1

Denaturation 9497 C30 sec

C30 sec

Elongation 72-80 C30-60 sec

Final elongation 75-80 C5-7 minx1

Stage 1

Stage 2

Stage 3

ÎOptimization done by applying

Example.

PCR reaction.

Optimization of PCR annealing temperature.

1. Start by applying the standard concentration of PCR component that work with majority of

2. Prepare a master mix that contains everything except the DNA template by multiplying the

3. Using special PCR tubes, distribute the master mix by pipetting ---µl to eahtube.

4. Add the DNA template for each template.

5. Centrifuge the tubes briefly.

6. Set the thermal cycling condition as following:

3. Try different 8 annealing temperatures depending on your primer pair Tm.

4. Set the final volume in the thermal cycler to be 50 µl.

5. Start PCR !!

Initial denaturation

All the rows have the

Denaturation

All the rows have the

Annealing

Each raw/column

Extension

All the rows have the