8. Complete this master mix table for 5 DNA samples. When...

8. Complete this master mix table for 5 DNA samples. When deciding how many reactions to calculate, be sure to include the necessary controls required to interpret the PCR result relative to contamination and functional reagents and equipment, and be sure to account for pipetting error. Show your work (attach extra sheet if necessary). (5 points) | Master Mix* | Conc. of Stock Solution | Final Conc. | ?L/Rxn (total per tube) | Number of Reactions | Total ?l Needed for master mix | |---|---|---|---|---|---| | PCR buffer (1) | 10X | 1X | | | | | dNTP mix (3) | 10 mM | 150 ?M | | | | | MgCl2 (4) | 45 mM | 9 mM | | | | | Forward Primer (5) | 20 ?M | 2 ?M | | | | | Reverse Primer (5) | 20 ?M | 2 ?M | | | | | Taq polymerase (6) | 6 U/?L | 0.09 U/?L | | | | | DNA | | | 1 ? L | | | | Water (2) | | | | | | | Total volume of the entire reaction (?Ls) | | | 20 ? L | | | 9. Draw out your pipetting scheme for your Master Mix and your PCR reactions. Draw a single 1.5 ml Eppendorf tube for your Master Mix (MM) and 0.2 ml PCR tubes for each of your PCR reactions. Label your drawings, indicating what each tube contains (e.g. "39 ul of MM and 1 ul of DNA"). (4 points)

Transcript

00:01 So you're given this chart of master mix and complete the table for five dna samples.

00:07 So when deciding how many reactions to calculate, be sure to include the necessary control.

00:14 So i assume that it will be negative control, which will eliminate all that contamination.

00:20 So that negative control will have all the component of a master mix without the dna and the positive control, which is to make sure to function or, reagent and equipment are working, this is going to be included dna that we know for sure it would work.

00:38 So show your work so that, you know, attach extra sheet if it's necessary.

00:46 So let's take a look for the first reaction.

00:51 So you can tell that there are several components, so you have to start from somewhere, so pcr buffer, let's say.

00:58 So we can actually use the very important formula down here.

01:04 Let me write down.

01:05 C1 times v1 equals c2 times v2.

01:10 C1 in this case is going to be stock concentration and v1 is the stock volume.

01:24 C2, final concentration, final volume v2.

01:36 Alright, now let's start with the first one, the pcr.

01:41 Buffer so we know that it start out with a stock concentration of 10x final 1x and we know the volume of each reaction is 20 so c1 10x and v1 is what we want to find out c2 is 1x and v2 is 20 mic liter so we can deduce that v1 is 20 times 10, divided by 10 equals 2 mic liter.

02:25 So then we put this in here.

02:28 We need 2 micliter of pcr buffer.

02:32 So use the same formula we can calculate the second component, dntp.

02:38 So dnt has 10 millimolar to begin with and 150 micromolar as a final concentration.

02:48 So dntp, so 10 millimolar times v1 equals 150 micromolar times 20 micliter.

03:07 Now one thing you have to first of all do is to convert the unit.

03:14 So one is millimolar, the other is micromolar.

03:18 So we want to make sure the concentration they have the same unit.

03:22 So 150 micromolar actually equals 0 .15 millimolar.

03:30 And this is because 1 millimolar equals 1 ,000 micromolar.

03:37 So now we convert this into both into millimolar.

03:42 So v1 is 0 .15 millimolar times 20 divide by 10.

03:54 Millimolar.

03:57 So in this case v1 equals 0 .3 microliter.

04:03 So make sure the volume has the same volume as the 20 mic liter same unit .3 mic liter.

04:13 The next item, magnesium chloride, this is .3.

04:21 Magnetium chloride start out with 45 millimolar and end with 9 millimolar.

04:29 So magnesium 45 millimolar times v1 equals 9mmolar times 20 micliter.

04:45 So again the same way, now these two are concentrations to have the same unit.

04:50 So all you have to do is v1, 9 times 20 divide by 45, you end up having 4 microliter, same unit.

05:09 Okay, the next one, forward and reverse primer.

05:11 They were actually the same thing.

05:13 So let's do one of it.

05:16 So the forward primer start out with 20 mic liter and end up with 2 micriter primers.

05:29 So 20, is it minimal or micrliter? i have to go micromolar.

05:35 Okay.

05:36 Times v1 equals 2 micromolar times 20 so v1 equals 2 micrometer divide by 20 so you end up having again 2 mic liter all right so 2 micliter so 2 micliter and 2 micliter so 2 micliter here now the tech polymerase okay so you have six unit per micleiter to begin with b1 equals 0 .0 9 unit per mic liter times 20 micliter v1 equals 0 .09 times 20 divide by 6.

07:00 Okay, so this gives us 0 .3 again, mic liter.

07:19 So i have filled in all the volume for each component in one reaction right here.

07:27 So this is per reaction.

07:29 So you add them up, and then the rest of the volume here is going to be water.

07:32 So you add 2 plus 0 .3 plus 4 plus 2 plus 2 plus 0 .3 plus 1 microliter of dna is 11 .6.

07:48 So now you know that the rest of the component is 11 .6.

07:54 20 minus 11 .6 equals 8 .4 micliter.

08:02 So the water down here, remember, is total of 20 minus rest, which in this case, 8 .4.

08:19 Okay, so this is in one reaction.

08:22 Now, think about how many reactions that we have.

08:26 So according to the question, we have five dna sample, and then you have one negative control.

08:30 As i mentioned, the negative control should be anything but the dna, because you want to make sure that these reagents are clean.

08:41 And then the positive control means that you should mix all the component with a dna sample that guaranteed to work to make sure these reagent equipment work properly...

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