Question

2. After your frustration with tissue culture, you finally get your cells passaged and decide to set up your cDNA synthesis reaction, PCR, and agarose gel. You have extracted RNA from your cells, and now you need to proceed with the cDNA synthesis.

a. The first step is to determine the concentration of your RNA. You dilute your RNA 1:250, vortex it, move it to the cuvette, and run it on the spectrophotometer.   The spec tells you that your concentration of RNA is 22µg/mL with an A260 of 0.32 and an A280 of 0.2. What is your ACTUAL RNA concentration, and what is its purity?

b. After determining your actual concentration of RNA, you are ready to make your cDNA. The protocol calls for the following for a total of 20µL:

5x iScript Reaction Mix – 4µL

iScript reverse transcriptase – 1µL

Nuclease free water – x µL

RNA template (100fg to 1ug total RNA) – x µL

You decide to use 500ng (or 0.5µg) of RNA in this reaction. How many µL of your RNA do you need, and how much water will you put in the tube?

c. You make the cDNA and get ready to set up your PCR for CD-10. The Qiagen protocol is as follows to a total of 50µL:

cDNA – 2µL

MgCl (25mM) – 4µL

CD-10 Forward Primer (10nM) – 1µL

CD-10 Reverse Primer (10nM) – 1µL

                                                10x Buffer – 5µL

                                                Q Solution – 10µL

                                                dNTPs (25mM) – 2µL

                                                Taq Polymerase – 0.5µL

                                                Water – to 50µL

You have 2 samples plus a negative control that you need to test. How will you set up a master mix to make your pipetting more accurate and setting up this experiment go much faster than pipetting each individual component into 3 separate tubes? Remember to give yourself room for error.

d. While your PCR is running, you decide to go ahead and make your agarose gel. Your product size is expected to be 536bp. What percentage agarose gel do you need? How much agarose, and how much 1X TBE buffer to you need to accomplish this?

e. You pour your gel successfully, but use the last of the 1X TBE. You need more TBE to run the gel, but all we have is 50X TBE. You decide to make 3L of 1X TBE from the 50X TBE so that there is plenty for others (remembering your frustration from tissue culture and no PBS earlier in the day). How much 50X TBE do you need, and how much water do you need to make 3L?

3. Making Solutions:

a. You need 100mLs of a 5M solution of CaCl₂. The MW of CaCl₂ is 111 grams/mol. How many grams of CaCl₂ do you need?

b. You want 25mLs of the following solution: 0.5M CaCl₂ and 1M MgSO₄. You have 5M CaCl₂ and 2.5M MgSO₄. How much of each do you add to make your final solution?

4. Many times, you will be using someone else’s lab notebook to formulate your own protocols. You will notice in some lab notebooks that the author will refer to nearly everything in volume rather than concentration. For example, it may say, “add 5μL DNA to the PCR tube” instead of “add 500ng DNA to the PCR tube.” Which is more accurate? Why?

Answer 1

2 a. An A260 reading of 1.0 is equivalent to ~40 µg/ml single-stranded RNA.The A260/A280 ratio is used to assess RNA purity. An A260/A280 ratio of 1.8-2.1 is indicative of highly purified RNA. The reading at 260 nm allows calculation of the concentration of nucleic acid, while the reading at 280 nm gives the amount of protein in the sample.

A260= 0.32; A280= 0.2

1OD at A260= 40mug/ml

So 0.32 OD at A260= x

Solving for x= 0.32 x 40/1=12.8 mug/ml is the concentration of the 1:250 diluted RNA sample

So the concentration of the original sample is 12.8mug/ml x 250=3200mug/ml or 3.2mg/ml.

The purity is checked by A260/A280= 0.32/0.2=1.6. This is lower than 1.8 indicating that the RNA sample has low purity.

2b. Concentration of original RNA sample= 3.2mg/ml or 3200 mug/ ml or 3200 mug in 1000mul

So to get 0.5mug;

3200-1000

0.5-x

Cross multiply and solve for X

x=0.5x 1000/3200=0.156mul of the original sample needs to be pippetted. This is a very small volume to pippete and can result in error. So first dilute the original sample 1:10. That is take 2mul of original sample and add 18mul water. This will make it 320mug/ml. Then we can take 1.56mul of the RNA sample and 13.44 mul of nuclease free water to the reaction mixture to make a total volume of 20mul.

c. First make a master mix as follows without cDNA for 4 tubes to allow room for error.

MgCl (25mM) – 4µLx 4=16µL

CD-10 Forward Primer (10nM) – 1µLx 4= 4µL

CD-10 Reverse Primer (10nM) – 1µLx 4=4µL

                                                10x Buffer – 5µLx 4=20µL

                                                Q Solution – 10µLx 4=40µL

                                                dNTPs (25mM) – 2µLx 4=8µL

                                                Taq Polymerase – 0.5µL x4=2mul

                                                Water – to 24.5µLx 4=98mul

Mix thoroughly and distribute 48mul in each of 3 tubes. Add 2 mul each of your cDNA samples and of your negative cDNA control to the three tubes and mix separately.

d A 1% agarose gel will be sufficient. You will need 0.3g agarose in 30ml 1X TBE.

e M1V1=M2 V2

50 x x= 1 x 3000ml

x=3000/50= 60 ml of 50X TBE plus 2940ml of water to make 3000ml or 3 L of 1X TBE

3a. No of moles= mass of a substance / molecular weight

Molarity= No.of moles/volume in litres=(mass of a substance / molecular weight)/volume in litres

Substituting in the above equation

5M= (x/111)/0.1L

5= x/ 11.1

x= 5 x 11.1= 55.5 g

You will need 55.5g of CaCl2  to make 100mLs of a 5M solution.

3b.The 25ml solution must contain both 0.5M CaCl2 and 1M MgSO4.

We use the formula M1V1=M2V2 to calculate the volumes of each

For 0.5M CaCl2;

5M x V= 0.5M x 25ml

V=0.5 x 25/5= 2.5ml of 5M CaCl2

For 1M MgSO4;

2.5M x V= 1 x 25

V= 10ml of 2.5 M MgSO4

So 2.5 ml of 5M CaCl2 + 10 ml of 2.5M MgSO4 +12.5ml of water (to make up the volume to 25ml) are required to make 25 ml of 0.5M CaCl2 and 1M MgSO4.

4. It is more accurate to use concentration and adjust he volume according to the required concentration for the reaction. If for example a PCR reaction requires 100ng of DNA and you have 10ng/mul of ur DNA sample u can add 10mul to the PCR reaction. Specifying volume is of no use as the concentration of DNA in that volume is not known and we may end up adding too little or too much DNA.