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1. During which step(s) of the PCR cycle are phosphodiester bonds being formed?
a. Denaturation
b. Annealing
c. Extension
d. During both annealing and extension

2. Estimate the Tm of the following primer using the method in this handout. (One easy method to
estimate the Tm of a primer is to count the number of G and C residues and multiply that
by 4°C then count the number of A and T residues and multiply that by 2°C then add the
two numbers.)
5’ TTATGCATGACTAATAGAA 3’

3. How many µl of the pGLO plasmid will you need for a PCR reaction if you need 500ng and the plasmid concentration is 0.05µg/µl?

4. What will the final concentration of the primer be if you add 1 µl of a 10µM stock in a reaction with a final volume of 25 µl?

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Jamar Ferry
Jamar FerryLv2
28 Sep 2019

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Related questions

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?

2

a) You also wish to synthesize a degenerate oligonucleotide primer that could be used eventually to clone DNA that encodes your mutant protein. Design this degenerate oligo, keeping in mind the rules we talked about in class. More information about designing degenerate primers and primers in general are located on the following page.

Write out the sequence of this oligo here

b) If you synthesized the above oligonucleotide primer, how many different oligos would be present in the mixture?

RULES TO FOLLOW for creating primers (Modified from the Thermo Fisher web site):

Designing degenerate primers:

Write out your amino acid sequence, label amino and carboxy termini

For each amino acid, use the genetic code to predict the various nucleic acid codons that can encode this amino acid. For all but Met, you should have redundancy present.

Now you need think about 5’ and 3’ considerations, and keep in mind that DNA within genes is double-stranded. It is helpful to keep in mind that the 5’ end of a gene corresponds to the amino terminus of a protein. Using 5’ and 3’ labels write out a single strand DNA sequence corresponding to the your amino seq of interest. You may choose to start with only possible codon for each amino acid, or you can do all possibilities using the notation I showed you in class to account for the wobble position of codons.

Now make your DNA double stranded by filling in the opposite DNA strand; label the 5’ and 3’ ends of this second strand and make sure you have an anti-parallel arrangement of DNA strands.

If you haven’t already, now consider the redundancy present on both strands, and make sure you have indicated sequences that account for all possible redundancies.

Realize that when a researched synthesizes a degenerate oligo that the final synthesis contains oligos with every substitution possible. The amount of degeneracy is defined by the number of different primer combinations in the mix. You can determine the degree of degeneracy by multiplying the number of changes present at each position together. If a position has no degeneracy then you multiple by 1 for that position.

Keep in mind that the trade-off between primer specificity and efficiency can be modified by altering the degeneracy of the primer. For example, the more degenerate the primers, the less specific annealing will be; however, decreased degeneracy will allow more potential to identify unknown variants. Anything over 1024 fold degeneracy is at the upper limit of potential usefulness.

Designing Primers:

Good primer design is essential for a successful PCR reaction. There are many factors to take into account when designing the optimal primers for your gene of interest. This information is taken from the Fisher Scientific website and are used when actually designing an experiment. Here are some tips to consider when designing primers.

In general, a length of 18 nucleotides is the minimum necessary for efficient primer binding.

Try to make the melting temperature (Tmm) of the primers between 65°C and 75°C, and within 5°C of each other.

If the Tmm of your primer is very low, try to find a sequence with more GC content, or extend the length of the primer a little.

Aim for the GC content to be between 40 and 60%, with the 3' of a primer ending in C or G to promote binding. This is called a GC clamp.

Typically, 3 to 4 nucleotides are added 5’ of the restriction enzyme site in the primer to allow for efficient cutting. Restriction enzymes are ENDO nucleases, so they cut better when their recognition site is not on the end of DNA.

Try to avoid regions of secondary structure, and have a balanced distribution of GC-rich and AT-rich domains.

Avoid intra-primer homology (more than 3 bases that complement within the primer) or inter-primer homology (forward and reverse primers having complementary sequences). These circumstances can lead to self-dimers or primer-dimers instead of annealing to the desired DNA sequences.


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