Calculation of Taq Polymerase - (Aug/15/2014 )

I am having trouble in figuring out the right amount of Taq polymerase required for PCR (conventional/uniplex)

I have Taq Polymerase (3U/μl) 1000 units supplied by the manufacturer. I want to use it at a final concentration of 1.25 U

To test out I want to run a single reaction with minimum volume of 10 μl.

The concentration of Polymerase is 3U/µl, this means 1 µl contains (1/3) 0.33 Units.

Normally a concentration of 1.25 U is used in final volume of 50 µl.

Therefore the volume used is (1.25/3) 0.416 µl for a final volume of 50 µl

Hence, calculating for 10 µl final volume, the polymerase should be (0.416 x 10/50) 0.08 µl

This is how I have modified my mixture:

10x PCR buffer: 1 µl

0.2 mM dNTP: 0.2 µl

0.1 µM F' primer: 0.1 µl

0.1 µM R' primer: 0.1 µl

Taq pol: 0.08µl

50ng DNA: 0.4 µl

Water: 8.12µl

----------------

Total: 10 µl

I am not getting any product after PCR. Where am I wrong?

-medimicro-

You are wrong here:

medimicro on Fri Aug 15 11:14:28 2014 said:

The concentration of Polymerase is 3U/µl, this means 1 µl contains (1/3) 0.33 Units.

But, you got the right answer in the calculation anyway (though you rounded 0.4166667 to 0.416 instead of 0.417...)

The reasons why you didn't get a product could be many - what sort of DNA was it (plasmid? genomic? plant? eukaryotic? bacterial?...etc) How was it extracted? Has it worked in PCR before?

Annealing temp?

Has this reaction worked before on a different template?

Did you mix properly?

Try diluting the DNA 1:10, 1:100, 1:1000 and see if it works (inhibitors in DNA prep).

Try scaling the reaction up to 20 or 50 ul.

-bob1-

Thanks for the reply.

Generally, 1.25U of polymerase is taken for 50µl final solution. If the final volume is scaled down to 10µl, how many units of polymerase should be taken?

The DNA is extracted from bacterial colonies by boiling. I have got wonderful results with 10 µl volume using reagents from a different company. Earlier, I had used polymerase that was 5U/µl

-medimicro-

You can't effectively measure 0.08 ul of taq (glycerol has a very high surface tension and is sticky, also micropipettes can't measure below 0.1 ul, and even at 0.5 ul, the measurements are suspect), you would be better off diluting the taq in something then adding it so as to get the correct amount.

I would still add a bit more taq than you are using, probably go for 1 U per reaction.

-bob1-