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18 Dec 2019

A genomic library is made for a haploid organis with a genome of3 X 10^9 bp. If you want to clone 5 genome equivalent into a vectorlibrary that carries 20 kb per contruct, how mny individual clonedo you need to make?

The correct answer is 7.5 X 10^5. But how do they get thisanswer?

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Trinidad Tremblay
Trinidad TremblayLv2
31 Dec 2019
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Related questions

Recently I have found myself in a discussion with a guy thatisn't a creationist, but denies evolution. He cites several studiesthat show problems like this one, but I do not have the knowledgeto refute it properly. Whats the problem with the following:

One problem is that population genetics shows us evolutiondestroys functional sequences much faster than it creates them inany large-genome animal with low reproductive rates, becausedeleterious mutations arrive faster than selection can remove them.This would include just about all mammals, reptiles, and birds--anyof our would-be ancestors over the last 300m years.

Specifically, humans get 60 to 160 mutations per generation,10-20+% of our genome is sensitive to substitution, and this givesus 6-32 deleterious mutations per offspring (most slight). Evenwith the low estimate of 6, every child is less fit than theirparents and natural (or even artificial) selection can only choosethe least degenerate each generation. For every generation where amutation can be selected, random mutations destroy 5-32+ previouslyselected. Beneficial mutations like lactose tolerance (a jammedswitch) take 1000s of years to appear and fixate.

If you know the genome size, mutation rate, and reproductiverate, you can calculate at what rate deleterious mutations arrivefaster than they can be removed:

"It has been estimated that there are as many as 100 newmutations in the genome of each individual human. If even a smallfraction of these mutations are deleterious and removed byselection, it is difficult to explain how human populations couldhave survived. If the effects of mutations act in a multiplicativemanner, the proportion of individuals that become selectivelyeliminated from the population (proportion of `genetic deaths') is1-e^-U, where U is the deleterious mutation rate per diploid, so ahigh rate of deleterious mutation (U>>1) is paradoxical in aspecies with a low reproductive rate." High genomic deleteriousmutation rates in hominids, (Nature, 1999)

They worry about a deleterious rate of U much greater than 1 asbeing prohibitively high and we're talking about it being 6-32+,since we now know the functional genome is much larger than wethought it was in 1999. Taking their Poisson probabilitydistribution and using U=6, that means 1-e^-6 = 99.752% of thepopulation will have to be selected away each generation for onelucky enough to have no deleterious mutations. For two to surviveand maintain constant population size that would require on average806 offspring per female--impossible for most mammals, birds, andreptiles. Actually much more since natural selection is notomnisciently efficient.

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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