Calculations for PCR Lab. Help! DNA Template - 895.4 ng/ul Forward Primer -100 u

Question

Calculations for PCR Lab. Help!

DNA Template - 895.4 ng/ul Forward Primer -100 uM Reverse Primer -100 uM dNTP Mix -10 mM MgCI2 - 25 mM Buffer - 5X Polymerase - 5 units/ul Total Reaction Volume will be 50 ul Dilute both primers to 10 uM concentration (make 50 ul) Final primer concentration in reaction is 1 uM. Calculate how much primer to use. DNA Template Dilute DNA to 100 ng/ul, make 15 ul How much diluted DNA is needed to have 300 ng/reaction? Final dNTP mix concentration of 0.2 mM Final MgCI2 concentration of 2

Explanation / Answer

In all the calculations, use the formula, N1V1=N2V2 where the left hand side represents the values for stock solution, and Right hand side represents the values for working solution.

For primer concentration,

N1V1=N2V2

100 *V1 = 10*50 ; V1= 5 microlt. Dissolve 5 microlt. stock solution in 45 microlt. distilled water to make 50 microlt. of 10micro mole primer solution.

Now, we want the final primer concentration in 50microlt. final solution should be 1 micromole.

10 *V1 = 1 * 50; V1= 5 microlt. So, 5 microlitre of forward and 5 microlt. of reverse primer should be added to the final solution.

DNA TEMPLATE:-

Stock template has concentration 895.4ng/microlt. And we have to make it 15 microlt. of 100ng/microlt.

895.4 *V1= 100 *15 ; V1= 1.67 microlt. (Mix 1.67 microlt. of stock in 13.33 microlt. of distilled water, to make the above concentration).

For final DNA template we need to know how many PCR reactions will be there.

dNTP

dNTP stock = 10mM and working solution required is 0.2mM

So, 10 * V1 = 0.2 *50 ; V1 = 1 microlt. (one microlt . of dNTP stock solution should be added to the final reaction mix.)

For MgCl2

25 *V1 = 2 *50; V1 = 4 microlt (4 microlt. of MgCl2 should be added to final reaction mix)

for buffer;

5*V1= 1*50; V1= 10 microlt.

For polymerase: we need to know how many reactions /pcr cycles will be there.

Forward primer = 5 microlt.

reverse primer = 5 microlt.

dNTP= 1 microlt.

MgCl2 = 4 microlt.

5X buffer = 10 microlt.

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