10. Complete this Master Mix table for 3 DNA samples, a...

10. Complete this Master Mix table for 3 DNA samples, a positive control, negative control, and an extra reaction for pipetting error. Show your work. (4 pts) Master Mix | Conc. Of Stock solution | Final Conc. | L/Rxn (total per tube) | Number of Reactions | Total L needed for master mix ---|---|---|---|---|--- PCR buffer | 20X | 1X | | | Water | | | | | dNTP mix | 100 mM | 100 M | | | MgCl2 | 25 mM | 2.5 mM | | | F Primer | 10 M | 0.1 M | | | R Primer | 10 M | 0.1 M | | | Taq polymerase | 5 U/L | 0.05 U/L | | | DNA | | | 3.5 L | | Total volume of the entire reaction (L) | | | 60 L | |

Transcript

00:01 Hello students, in this question we need to make up the final volume or the total volume to 25 microlitre and for that we need to calculate the other components.

00:13 So, the formula for that will be c1 into v1 is equal to c2 into v2.

00:21 So, this formula we need to remember and should call this formula the major formula to calculate all the other components, you should remember this formula.

00:37 So, c1 is the stock concentration, v1 is the volume of the stock, c2 is the final concentration and v2 is the volume of the reaction.

00:46 Now first of all there is distilled water, we need to calculate this later.

00:54 In the next part we need to calculate the 5x buffer that should be added to make up the final concentration of 25 microlitre.

01:03 So, in that case the c1 is 5x, we need to calculate the v1, c2 is 1x and v2 is 25 microlitre.

01:18 So, v1 in this case will become 25 into 1 divided by 5 that should be 5 microlitre.

01:29 So, 5 microlitre of tac buffer should be added to make up the final concentration or the final volume to 25 microlitre.

01:38 In the next case mgcl2 of 50 millimolar concentration should be made by c1 is 50 millimolar, we need to calculate the v1, c2 is 5 millimolar and the final concentration or the final volume is 25.

01:59 So, in this case 5 into 25 divided by 50, so v1 will become 2 .5 microlitre in this scenario.

02:13 So, this is the concentration of mgcl2 that should be added to make up the final concentration or the final volume of 25 microlitre.

02:28 Next to calculate dntps or dntp mix of 10 millimolar stock, we need to calculate the v1, c2 is 0 .2 millimolar and the final volume is 25.

02:46 So, v1 in this case is 0 .5 microlitre.

02:51 Now, the next part is forward primer.

03:00 So, forward primer of 5 millimolar stock should be calculated by the formula.

03:09 So, here 5 is the c1 concentration, we need to calculate v1, c2 is 0 .5 micromolar and 25 is the concentration...

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