Question

Prepare a fragment of DNA to be cloned by PCR by preparing the oligonucleotides received through dilutions. The DNA to be used as a template is in a 50 ng/ul solution. The protocol is as follows:

VOLUME TO ADD FINAL CONCENTRATION 0.5 UM 0.5 M REAGENT 10 PM Forward Primer 10 PM Reverse Primer Thermoestable pol Master mix 2x Template DNA water 1x 100 ng.. Total volume 25 ul Information about the primers: Forward primer: 29.3 nmoles - 220 ug - molecular weight 7,586 Reverse primer: 33.1 nmoles - 180 g-molecular weight 5,490 1. How much water you need to add to dissolve them to 100 MM solutions 2. How do you prepare the primers 10 M solutions? 3. Complete the volume values in the table

Answer 1

Molaority = mass/molecular mass * 1/volume in ul

1 - For forward primer

Molarity = 100uM

mass = 220ug

Molecular mass = 7586.

Volume = need to find.

100 = 220 / 7586 * 1/ volume

Volume = 220/100 * 7586

= 290 ul.

So we need to dissolve forward primer to in 290 ul to make its 100uM solution

For reverse primer

Molarity = 100uM

mass = 180ug

Molecular mass = 5490

Volume = need to find.

100 = 180 / 5490 * 1/ volume

Volume = 180/100 * 5490

= 328 ul.

So we need to dissolve forward primer to in 328 ul to make its 100uM solution

B- To make 10uM solution we need to take 10ul form the primer solution and add 90 ul water to make final volume 100ul

C - C1V1= C2V2

C1 = 10uM

V1 = need to find out

V2 = 25ul

C2 = 0.5uM

10*V1 = 25*0.5

V1 = 12.5/10 = 0.125 ul.

We need to add 25/2 = 12.5 ul of Mastermix.

DNA concentration is 50ng/ul.

We need 100ul for the reaction.

So we need to add 100/50 = 2 ul.

The volume of water = 25- volume of all reagants.

volume of all reagants. = 12.5+0.125+0.125+2 = 15ul

So the volume of water = 25-15 = 10ul

VOLUME TO ADD 0.125 ul 0.12501 REAGENT 10 PM Forward Primer 10 PM Reverse Primer Thermoestable pol Master mix 2x Template DNA water FINAL CONCENTRATION 0.5 M 0.5 M 100 ng.. 12.5ul Zul 10 ul Total volume 25 ul