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Calculate the following for a 200 µl DNA preparation that a 10 µl aliquot when diluted 100-fold yielded an OD260 of 0.163 and OD280 of 0.087. Concentration – Yield – Purity ratio – Would you consider this to be a quality preparation? Why or why not?

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Hubert Koch
Hubert KochLv2
29 Sep 2019

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Related questions

The second PCR you perform requires that only 3-10ng of PCR product be used for ideal results. This, of course, must be paired with careful lab technique, such as keeping the sequencing PCR mix ice-cold and not contaminating the reaction with outside sources of DNA such as that on your fingertips. The following steps will take you through a proper dilution of the PCR product so that you get the appropriate concentration of DNA for your sequencing reaction. Remember: you cannot pipet 0.5 µl or 1 µl with a P20 micropipettor. The lowest volume you can pipet is 2 µl. Attempting to set it lower than that can break the equipment!

As per the instructions on page 35 of your manual, you need to add:

4 µl of [PCR product + water] that has a total of 3-10ng of DNA

to the 0.2ml PCR tube that already contains 6 µl of the Big Dye Master Mix (=Sanger Sequencing PCR Mix) to get a 10 µl reaction.

If you need 3-10 ng/4 µl, what is the DNA concentration range per 1 µl required?

Many of you will have a band equally bright to the 500bp SS band, so go back to #3 in Step 1 and record that amount here:

______________ ng DNA/ µl PCR product

How many times higher is the concentration of DNA in #7 as compared to the concentration required per microliter (#6)? [For example, if you had a sample that had 1500ng DNA/µl and you needed ~125ng DNA/µl, your sample would be 12x higher than it should be.]

To get DNA at the volume and concentration you need, use the # calculated in #8 to determine your proportion of sample:water. This diluted DNA would need to be mixed in an empty, separate tube and can be at any volume so long as the ratio is correct (though keep in mind you only have ~40 µl of PCR product remaining).

Using the example from #8, if we had a sample that was 12x more concentrated than required, we would make a dilution that was 1/12 sample and 11/12 water. This would be a 1:11 ratio. Since the minimum volume we can accurately pipet is 2µl, we could make a tube with 2µl sample (=3000ng DNA) and 22µl water = total of 3000ng DNA/24µl water = 125ng/µl. 4µl of this diluted DNA could then be pipetted out and added to the desired reaction.

Work with your partner to determine how to make a DNA dilution that would be appropriate for sequencing, yielding at least 4µl total volume containing only 3-10ng DNA. Again, your minimum pipetting volume is 2µl.

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?

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.

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?

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?

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.

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?

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?

Making Solutions:

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?

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?

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?

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