Hi Aalaa

you can prepare a master mix by mixing PCR component as following:

(For 25 microlitr reaction)

buffe 10X=2.5 micro litr

dNTPs(10mM) =0.5 microlitr

MgCl2(50mM) = 0.75-1 microlitr

primer forward(10 picolitr)=0.8-1.5

primer reverse(10 picolitr)=0.8-1.5

Taq polymerase (5U/microlitr)= 0.15-0.25 microlitr

RNase-free water=variable

Note: keep the PCR tube on ice( when working).

master mix is ready.

Then mix the master mix thoroughly, and then add template(DNA or cDNA =1-2.5 microlitr)

Hi Aalaa

you can prepare a master mix by mixing PCR component as following:

(For 25 microlitr reaction)

buffe 10X=2.5 micro litr

dNTPs(10mM) =0.5 microlitr

MgCl2(50mM) = 0.75-1 microlitr

primer forward(10 picolitr)=0.8-1.5

primer reverse(10 picolitr)=0.8-1.5

Taq polymerase (5U/microlitr)= 0.15-0.25 microlitr

RNase-free water=variable

Note: keep the PCR tube on ice( when working).

master mix is ready.

Then mix the master mix thoroughly, and then add template(DNA or cDNA =1-2.5 microlitr)

Aala,

If you're making your own, depending on the mix you're using and the concentration of your extracted DNA it should look something like this:

Preparing a 20uL (TOTAL including DNA)

1. Buffer mix (if you're using a 10x dilute it 10 times etc...) ml

2. Forward primer 1uL

3.Reverse primer 1uL

4.Taq polymerase 0.5-1uL

5. Water

6. Template DNA 1mg (again the volume will depend on the DNA concentration that you have) X uL

Calculate these volumes up until ingredient 5, which is the DNA. So if you require 2uL of DNA solution to get 1ug of DNA ingredients 1 through 5 should have a total of (20-X)uL.

IF you need 20 reactions prepare 10% more master mix due to pipette inaccuracies and aliquot it in pcr reaction tubes. THEN add the amount of DNA necessary to add 1ug of DNA AND have a total volume of 20uL.

Run your PCR. In case you need a bigger or smaller reaction volume adjust accordingly the amount of water.

This is just a guideline and you might need to adjust it,

Best of luck

Thanx for reply

my master mix is 5x conc ready to use solution how can i mix it with my DNA and primers

Hi Aalaa

If you are making 20 ul reaction mixture, then add 4 ul of your master mix and then 1-1 ul of your forward and reverse primers (10 pmole/ul) and 1 ul of DNA. rest of the volume is d/w.

Hi Aalaa, with a 5x enzyme mix, for a 20 ul reaction you would add 4 ul of your mix, 1-2 ul of your forward and reverse primers (final concentration 250 - 300 nM final initially). I would strongly suggest to add a higher volume of DNA or cDNA than 1 ul as I find that there is too much error in technical replicates using this volume. So I would suggest to add about 4-5 ul of DNA. Then you can complete with water your master mix to get 20 ul.

The fact that you "dilute" your DNA won't matter in the end as you'll be adding less water to your master mix, so in the end, the amount of DNA will be the same. In other words, if you add 4 ul of DNA instead of 1 ul, dilute your original DNA 4x with water and then add 4 ul of this DNA to your PCR reaction. Hope this make sense for you.

Best,

Thanx all

1-2 ul of your forward and reverse primers mean 1-2 ul of forward +reverse primers

or

1-2 ul forward and 1-2ul reverse primers?

1-2uL of your forward AND 1-2uL of your reverse.

Total 2-4uL

Thanks very much

Dear Aalaa,

PCR reaction contained 50–100 ng template DNA,

master mix by mixing PCR component as following:

250 nM forward primer,

250 nM reverse primer,

0.2 mM dNTPs,

2.5 μl PCR buffer (10X),

1U Taq DNA polymerase in a total volume of 25 μl.

What volume of  forward and reverse primer is need for 10 micro liter master mix?

The volume of Primers can be calculated by usng formula ViCi=V2C2 .

Thank you very much, if you explain the equation that will be great

If we increase amount of reagent (dNTP,DMSO,F.primer,R.primer) then what happen in PCR reasult?

Thank you for your answer

for every DNA template its different, need to test

from my experience up to 5 uM of F and R primer would work fine. however, 0.1 - 1 uM is recommended. uM is (micro molar)

Volume 10 microliter 25 microliters 50 microliters

Components

Buffer 10X 1 ul 2.5 ul 5 ul

dNTPs (10mM) 0.2 ul 0.5 ul 1 ul

MgCl2 (50mM) 0.3 – 0.4 ul 0.75 – 1 ul 1.5 – 2 ul

F- Primer (10 picolitr) 0.32 – 0.6 ul 0.8 – 1.5 ul 1.6 – 3 ul

R- Primer (10 picolitr) 0.32 – 0.6 ul 0.8 – 1.5 ul 1.6 – 3 ul

Taq polymerase

(5U/microlitr) 0.06 – 0.1 ul 0.15 – 0.25 ul 0.3 – 0.5 ul

RNase free water (6 – 7.5 ul) (15 – 19 ul) (30 – 39 ul)

DNA template 0.4 – 1 ul 1 – 2.5 ul 2 – 5 ul

Total Volume 10 ul 25 ul 50 ul

you can prepare a master mix by mixing PCR component as following:

(For 25 microlitr reaction)

buffe 10X=2.5 micro litr

dNTPs(10mM) =0.5 microlitr

MgCl2(50mM) = 0.75-1 microlitr

primer forward(10 picolitr)=0.8-1.5

primer reverse(10 picolitr)=0.8-1.5

Taq polymerase (5U/microlitr)= 0.15-0.25 microlitr

RNase-free water=variable

Note: keep the PCR tube on ice(when working).

master mix is ready.

Then mix the master mix thoroughly, and then add template (DNA or cDNA =1-2.5 microlitr)

All quantities are in Microlitres

you can prepare a master mix by mixing PCR component as following:

(For 25 micro liter reaction)

buffe 10X=2.5 micro litr

dNTPs(10mM) =0.5 microlitr

MgCl2(50mM) = 0.75-1 microlitr

primer forward(10 picolitr)=0.8-1.5

primer reverse(10 picolitr)=0.8-1.5

Taq polymerase (5U/microlitr)= 0.15-0.25 microlitr

RNase-free water=variable

you can prepare a master mix by mixing PCR component as following:

(For 25 micro liter reaction)

buffe 10X=2.5 micro litr

dNTPs(10mM) =0.5 microlitr

MgCl2(50mM) = 0.75-1 microlitr

primer forward(10 picolitr)=0.8-1.5

primer reverse(10 picolitr)=0.8-1.5

Taq polymerase (5U/microlitr)= 0.15-0.25 microlitr

RNase-free water=variable