PCR calculations Ali I.A.Gharip, BSc Biotech T.A
Description: PCR calculations Ali I.A.Gharip, BSc Biotech T.A The common volume of a PCR is10,25, 50, or 100μL. Although larger volumes are easier to pipet, they also use up a larger amount of reagents, which is less economical. All of the reaction
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slide1. PCR calculations Ali I.A.Gharip, BSc
Biotech T.A<br>
slide2. The common volume of a PCR is10,25, 50, or 100μL.
Although larger volumes are easier to pipet, they also use up a larger amount of reagents, which is less economical.
All of the reaction components can be mixed in together in a 0.5-mL PCR tube in any sequence except for the DNA polymerase, which should be added last.
It is recommended to mix all the components right before PCR cycling. Although it is not necessary to set up the PCR on ice, some published protocols recommend it.<br>
slide3. For each PCR, the following components are mixed together: with it's optimum ranges 1. Template DNA (1–500 ng).
2. Primers (0.05–1.0 μM).
3. Mg2+ (0.5–5mM).
4. dNTP (20–200μM each).
5. 1× PCR buffer: 1mM Tris-HCl and 5mM KCl.
6. DNA polymerase (0.5–2.5 U for each 50μL of PCR).<br>
slide4. The stock solution is: 1 ng/μL template DNA
50μM forward PCR primer
50μM reverse PCR primer
25mM MgCl2
2.5mM dNTPs
10× PCR buffer
5 U/μL Taq DNA polymerase<br>
slide5. Dilution equation: C1V1=C2V2
C1=original concentration (stock).
V1=original volume needed to dilute in the PCR final concentration.
C2=required concentration within the optimum ranges.
V2=PCR total volume.<br>
slide6. DNA template :<br>
slide7. Another example:<br>
slide8. Calculation of PCR primer:<br>
slide9. MgCl2:<br>
slide10. dNTPs<br>
slide11. PCR buffer:<br>
slide12. Taq DNA polymerase:<br>
slide14. 1μL of 1 ng/μL template DNA (final amount = 1 ng).added individually
Master mix for only one sample:
1μL of 50μM forward PCR primer (final concentration = 1μM).
1μL of 50μM reverse PCR primer (final concentration = 1μM).
5μL of 25mM MgCl2 (final concentration = 2.5mM).
4μL of 2.5mM dNTPs (final concentration = 200μM) each
or 4ul of 2.5mM dNTPs mix (final concentration = 200μM).
5μL of 10× PCR buffer (final concentration = 1×).
0.25μL of 5 U/μL Taq DNA polymerase (final amount = 1.25 U).<br>
slide15. calculate the H2O amount:<br>
slide16. Preparing a PCR final volume25μL Master Mix For 20 samples :<br>
slide17. Adding the PCR component individually is a lot of pipetting and each time you pipet you can introduce some error in the measurement. So we will use a way to minimize the pipetting. We will make a Master Mix.
The Master Mix should contain all the reagents needed for the PCR except the template DNA (which should always be added last to a PCR).<br>
slide18. We prepare master mix containing the primers with 23 fold more than aliquot
20 samples + 1 –ve control + 1 +ve control + 1 pipetting error =23
Or
We prepare master mix containing the primers with 55 fold more than aliquot
20 samples + 1 –ve control + 1 +ve control + 3 pipetting error =25
( so multiply the calculated volume minus template * 25)
20*25=500 μL ( for 22 tube)<br>
slide19. For PCR final volume25μL: 5μL of 50ng/μL template DNA (final amount = 10 ng).
1μL of 25μM forward PCR primer (final concentration = 1μM).
1μL of 25μM reverse PCR primer (final concentration = 1μM).
2.5μL of 25mM MgCl2 (final concentration = 2.5mM).
2.5μL of 2.5mM dNTPs (final concentration = 200μM)each or
2.5ul of 2.5mM dNTPs mix (final concentration = 200μM).
2.5μL of 10× PCR buffer (final concentration = 1×).
1μL of 5 U/μL Taq DNA polymerase (final amount = 5 U).<br>
slide20. 20 sample For PCR final volume25μL: 5μL of 50ng/μL template DNA (final amount = 10 ng) added individually
25*1μL of 25μM forward PCR primer (final concentration = 1μM).
25*1μL of 25 μM reverse PCR primer (final concentration = 1μM).
25*2.5μL of 25mM MgCl2 (final concentration = 2.5mM).
25*2.5μL of 2.5mM dNTPs (final concentration = 200μM)each or
25*2.5ul of 2.5mM dNTPs mix (final concentration = 200μM).
25*2.5μL of 10× PCR buffer (final concentration = 1×).
25*1μL of 5 U/μL Taq DNA polymerase (final amount = 5 U).<br>
slide21. 5μL of 50ng/μL template DNA (final amount = 10 ng) added individually
25μL of 25μM forward PCR primer (final concentration = 1μM).
25μL of 25μM reverse PCR primer (final concentration = 1μM).
62.5μL of 25mM MgCl2 (final concentration = 2.5mM).
62.5μL of 2.5mM dNTPs (final concentration = 200μM)each or
62.5ul of 2.5mM dNTPs mix (final concentration = 200μM).
62.5μL of 10× PCR buffer (final concentration = 1×).
25μL of 5 U/μL Taq DNA polymerase (final amount = 5 U).<br>
slide22. Total volume with dNTPs each (Avery one separate) except DNA template =25+25+62.5+4*62.5+62.5+25=450μL
total volume –total DNA =(25*25)-(25*5)=625-125=500
The amount of water=500-450=50dH2O
Total volume with dNTPs mix (all in one vial) except DNA template =25+25+62.5+62.5+62.5+25=262.5μL
total volume –total DNA =(25*25)-(25*5)=625-125=500μL
Then 500-262.5=237.5dH2O.<br>
slide23. Transfer 20μL into 22 tubes then discharge the 23rd tube (error tube) then add the template to every one tube , then the final volume is 25μL for each tube containing the template, then insert the tubes inside the PCR machine.
The aim of making more tube (error tube) is to;
Minimizes pipetting action.
Decrease pipetting error.
It is not easy collecting the final volume from reagent tube to PCR tube.<br>
slide25. All the best<br>
Biotech T.A<br>
slide2. The common volume of a PCR is10,25, 50, or 100μL.
Although larger volumes are easier to pipet, they also use up a larger amount of reagents, which is less economical.
All of the reaction components can be mixed in together in a 0.5-mL PCR tube in any sequence except for the DNA polymerase, which should be added last.
It is recommended to mix all the components right before PCR cycling. Although it is not necessary to set up the PCR on ice, some published protocols recommend it.<br>
slide3. For each PCR, the following components are mixed together: with it's optimum ranges 1. Template DNA (1–500 ng).
2. Primers (0.05–1.0 μM).
3. Mg2+ (0.5–5mM).
4. dNTP (20–200μM each).
5. 1× PCR buffer: 1mM Tris-HCl and 5mM KCl.
6. DNA polymerase (0.5–2.5 U for each 50μL of PCR).<br>
slide4. The stock solution is: 1 ng/μL template DNA
50μM forward PCR primer
50μM reverse PCR primer
25mM MgCl2
2.5mM dNTPs
10× PCR buffer
5 U/μL Taq DNA polymerase<br>
slide5. Dilution equation: C1V1=C2V2
C1=original concentration (stock).
V1=original volume needed to dilute in the PCR final concentration.
C2=required concentration within the optimum ranges.
V2=PCR total volume.<br>
slide6. DNA template :<br>
slide7. Another example:<br>
slide8. Calculation of PCR primer:<br>
slide9. MgCl2:<br>
slide10. dNTPs<br>
slide11. PCR buffer:<br>
slide12. Taq DNA polymerase:<br>
slide14. 1μL of 1 ng/μL template DNA (final amount = 1 ng).added individually
Master mix for only one sample:
1μL of 50μM forward PCR primer (final concentration = 1μM).
1μL of 50μM reverse PCR primer (final concentration = 1μM).
5μL of 25mM MgCl2 (final concentration = 2.5mM).
4μL of 2.5mM dNTPs (final concentration = 200μM) each
or 4ul of 2.5mM dNTPs mix (final concentration = 200μM).
5μL of 10× PCR buffer (final concentration = 1×).
0.25μL of 5 U/μL Taq DNA polymerase (final amount = 1.25 U).<br>
slide15. calculate the H2O amount:<br>
slide16. Preparing a PCR final volume25μL Master Mix For 20 samples :<br>
slide17. Adding the PCR component individually is a lot of pipetting and each time you pipet you can introduce some error in the measurement. So we will use a way to minimize the pipetting. We will make a Master Mix.
The Master Mix should contain all the reagents needed for the PCR except the template DNA (which should always be added last to a PCR).<br>
slide18. We prepare master mix containing the primers with 23 fold more than aliquot
20 samples + 1 –ve control + 1 +ve control + 1 pipetting error =23
Or
We prepare master mix containing the primers with 55 fold more than aliquot
20 samples + 1 –ve control + 1 +ve control + 3 pipetting error =25
( so multiply the calculated volume minus template * 25)
20*25=500 μL ( for 22 tube)<br>
slide19. For PCR final volume25μL: 5μL of 50ng/μL template DNA (final amount = 10 ng).
1μL of 25μM forward PCR primer (final concentration = 1μM).
1μL of 25μM reverse PCR primer (final concentration = 1μM).
2.5μL of 25mM MgCl2 (final concentration = 2.5mM).
2.5μL of 2.5mM dNTPs (final concentration = 200μM)each or
2.5ul of 2.5mM dNTPs mix (final concentration = 200μM).
2.5μL of 10× PCR buffer (final concentration = 1×).
1μL of 5 U/μL Taq DNA polymerase (final amount = 5 U).<br>
slide20. 20 sample For PCR final volume25μL: 5μL of 50ng/μL template DNA (final amount = 10 ng) added individually
25*1μL of 25μM forward PCR primer (final concentration = 1μM).
25*1μL of 25 μM reverse PCR primer (final concentration = 1μM).
25*2.5μL of 25mM MgCl2 (final concentration = 2.5mM).
25*2.5μL of 2.5mM dNTPs (final concentration = 200μM)each or
25*2.5ul of 2.5mM dNTPs mix (final concentration = 200μM).
25*2.5μL of 10× PCR buffer (final concentration = 1×).
25*1μL of 5 U/μL Taq DNA polymerase (final amount = 5 U).<br>
slide21. 5μL of 50ng/μL template DNA (final amount = 10 ng) added individually
25μL of 25μM forward PCR primer (final concentration = 1μM).
25μL of 25μM reverse PCR primer (final concentration = 1μM).
62.5μL of 25mM MgCl2 (final concentration = 2.5mM).
62.5μL of 2.5mM dNTPs (final concentration = 200μM)each or
62.5ul of 2.5mM dNTPs mix (final concentration = 200μM).
62.5μL of 10× PCR buffer (final concentration = 1×).
25μL of 5 U/μL Taq DNA polymerase (final amount = 5 U).<br>
slide22. Total volume with dNTPs each (Avery one separate) except DNA template =25+25+62.5+4*62.5+62.5+25=450μL
total volume –total DNA =(25*25)-(25*5)=625-125=500
The amount of water=500-450=50dH2O
Total volume with dNTPs mix (all in one vial) except DNA template =25+25+62.5+62.5+62.5+25=262.5μL
total volume –total DNA =(25*25)-(25*5)=625-125=500μL
Then 500-262.5=237.5dH2O.<br>
slide23. Transfer 20μL into 22 tubes then discharge the 23rd tube (error tube) then add the template to every one tube , then the final volume is 25μL for each tube containing the template, then insert the tubes inside the PCR machine.
The aim of making more tube (error tube) is to;
Minimizes pipetting action.
Decrease pipetting error.
It is not easy collecting the final volume from reagent tube to PCR tube.<br>
slide25. All the best<br>