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Friday, April 5, 2013

qPCR Calculations

qPCR or Real time PCR  has got several applications in diagnostics, forensic science and in many other biotechnology areas. The main advantage of PCR is its sensitivity even very small quantities of DNA can be detected using this method making it a powerful tool in the diagnostics and other areas.
Lets us look into some of the problem which beginners face while preparing master mix and setting up qPCR reactions.

Master mix Preparation for qPCR
PCR reaction mix also known as master mix is a combination of Buffer, Primers, Probe, Taq Polymerase, etc. it is prepared as for the no of reactions to be done. it is prepared in bulk and aliquoted into individual PCR tubes or PCR strips.

There are commercially available master mixes of various strengths like 5X, 2X, etc (Eg: 2X Universal Master Mix from takara), for a 10uL PCR reaction 5uL of the 2X master mix is required. (required volume / concentration of the master mix =10/2= 5uL).

Primer - Probe Dilution:
Primer is an oligoncleotide which binds specifically  to its complementary sequence in the target DNA. Primers are designed in such a way that it will bind only to particular region of the target. Primers can be custom synthesized depending on the sequence of the target DNA to be detected.
Custom synthesized primers are usually shipped to the customer as lyophilized powder along with a data sheet. Data sheet will have the information about the synthesis scale, yield, concentration, Optical Density readings, volume required to make 100uM solution.

Primer Reconstitution & Dilution
Primer stocks are maintained at 100uM concentration and the working stock will be of 10uM (Varies depending on the convenience of the person working).
A 100 micro Molar (uM) primer stock solution corresponds to 100 picomoles/uL (pmols/uL). one of the doubt i had when i started working on real time PCR how 100uM = 100pmols/uL, yes it is lets see how it is:
before entering into that just look at the basic conversions:
as we all know 1 Molar solution has 1 mole/Litre (Recollect the defintion of Molarity,"Molarity is defined as the no of moles per liter of a solution"). Eg: 1M NaOH solution has 1 mole of NaOH in 1 Litre.
1micro Molar solution will have 1 micromole / litre of a solution.

1uM = 1micromole /Litre or 1*10^6 picomoles/ Litre
= 1*10^6 pmols/1000mL
=1*10^6/10^3 pmols/mL
=1*10^3 pmols/mL ; (1mL = 1000uL) so,
=1*10^3 pmols/1000uL
=1*10^3 pmols /10^3 uL
=1 pmol/uL.

Similarlly 100uM primer solution will have 100pmols/uL of primer.

On adding required volume of TE Buffer or Sterile water to the lyophilized powder as mentioned in the primer data sheet, 100uM primer solution will be obtained and this Procedure is called as Primer Reconstitution. Reconstituted primers can be stored at -20 dC.

Primer Analysis
Primer once reconstituted needs to be analyzed by taking OD@260 in a spectrophotometer for its concentration. even though the supplier provides the information in the data sheet, it is good to have the concentration checked before proceeding with the reaction. lets see how to check the primer concentration in a spectrophotometer.

Dilute the reconstituted primer 100 fold to a volume of 1mL. [10uL of primer stock + 990uL of TE Buffer].
read absorbance at OD260. using Oligo Calc primer concentration can be easily calculated by inputting the primer sequence and the OD reading.
[Note: multiply the OD 260 reading into 100, as we have diluted it 100 fold].

concentration of primer

Another method of checking the primer is by running an Oligonucleotide PAGE, (Oligonucleotide PAGE protocol). Primer degradation can be found out by running the oligonucleotide PAGE.

Primer Dilution: Preparation of primer working stock
Once primer is reconstituted, working stock need to be prepared for using in PCR reaction. (some people use directly from the primer stock), generally working stock is maintained as 10uM solution. a ten fold dilution of the stock solution can be done to get a 10uM primer working stock solution.
Lets say you need a 100uL of 10uM primer working stock;
C1*V1 = C2*V2
100*V1  = 10*100
V1= 10uL.

You need to take 10uL of the 100uM stock and add 90uL of water to obtain 100uL of 10uM solution.

Probe Reconstitution and Dilution
Probe reconstitution and dilution is also done in same way as mentioned above only difference is probes will be coluored while primers are colourless.
Probe is reconstituted to 100uM and diluted 10 times to get 10uM working stock.

Taq Polymerase
Taq polymerase is the enzyme which helps in the synthesis of DNA copies from the template. there are two types of Taq Polymerase available wild Taq and Hot Start Taq (More on Hotstart Taq Polymerase and Types).
Taq polymerase is available in various concentrations , lets see one and find out how it is used in PCR reaction.
Eg: Hot Start Taq Polymersae 5U/uL
you need 2units for your 10uL PCR reaction, how many uL of  taq to use:
here one thing you need to keep in mind the taq stock has a concentration of 5U/uL and you need 2 units/10uL. Make both the concentration to same units
2U/10uL = 0.2U/uL
so C1*V1 = C2*V2
5U/uL*V1 = 0.2U/uL * 10
= 0.4uL
you need to take 0.4uL of 5U/uL of Taq to get 2U/10uL.

PCR reaction volumes are made up to the required volume by using sterile water. quality of water is important in PCR. Distilled water or sterile water free of DNase and RNases are recommended for usage.

Template is your DNA / RNA sample.

Oil, mineral oil is added to the PCR reaction tube inorder to prevent evaporation. it acts as a vapour barrier.In some of the mordern PCR machines oil addition to the PCR tube is not needed.

A general PCR Reaction compostition of 10uL PCR Reaction

2X Buffer                   - 5.0uL
10uM FOrward Primer - 0.3uL
10uM Reverse Primer - 0.3uL
Probe                          -  0.2uL
Water                        - 0.2uL
Template            - 4.0uL
Total                         - 10.0uL