Lab: RNA. Lab: RNA

In this lab, you will learn about the types of RNA molecules and you will study the details of transcription and translation.

Objectives:

Learn about the types of RNA molecules produced by cells.
Study the details of transcription and translation.

Time Requirements:

This lab should take two hours to complete.

RNA Lab Report

If a codon is mutated, say from GGU to CGU, is the same amino acid specified? _______

Using information from the course and online research, answer the questions.

Searching Websites:

Conduct a keyword search to find websites. To begin a keyword search, start by searching broad terms such as RNA, transcription, translation, or the scientist’s name. The resulting list of websites can give you a lot of information, but how do you know if it is reliable? Some basic guidelines can help you when you open a website and try to determine its reliability.

Always consider a site’s:

· Objectivity – Excessive expressions of emotion, opinions, and stereotyping are tip-offs that the information on a site may be biased.

· Ownership and contributors – Go to the Home or About page of the website and find out who sponsors and writes for the site. Look for contributors who have reliable credentials, such as “Harvey Jones, Professor, University of Wisconsin—Madison.”

· Writing style and mechanics – Check the grammar, spelling, and writing style on the site. Errors and awkwardness are signs of a nonprofessional website.

· Currency – Look for publication or copyright dates associated with the site; the more current the better.

· Links – What links does the site contain? A reliable website will offer links to other reliable websites, not to “junk” sites.

Keyword Search: RNA, transcription, translation, or the scientist’s name.

Scientific Analysis:

1. Scientists use observations and inferences for many discoveries and conclusions in genetics. What is the difference between an observation and an inference? Give an example of each.

2. If DNA from a cell is placed in a test tube containing the enzyme DNA polymerase and nucleotides, the result is new copies of the original DNA. This evidence was used to understand DNA replication (DNA passing information from one generation to the next). Write an explanation detailing why this clue represents solid data and is not an opinion or an inference.

3. In your own words, describe Crick’s Central Dogma. Explain why this is an inference.

Scientific Contributions

There were many scientists that contributed to decoding the genetic code. Explore the contributions of some scientists.

4. Describe Marshall Nirenberg’s contribution to determining the role of RNA in genetics.

5. Describe how Seymour Benzer’s phage experiments contributed to deciphering the genetic code.

6. Describe Seymour Brenner’s contribution to cracking the genetic code with respect to translation.

Replication, Transcription, and Translation

Place an x in the box for which process each item is a characteristic.

Characteristic	Replication	Transcription	Translation
1. RNA polymerase unwinds the DNA double helix.
2.  This is the first part of protein synthesis.
3. After the stop codon is reached, the protein is separated from the ribosome.
4. Amino acids sequences are made from RNA sequences.
5. A copy of RNA is made from DNA.
6. Peptide bonds form between amino acids.
7. Uracil hydrogen bonds with adenine.
8. tRNA brings amino acids to the ribosome.
9. Copies of the original DNA are made.
10.  Thymine hydrogen bonds with adenine.

 

7. Use the table and your research to compare and contrast transcription and translation.

8. In your own words, explain the function of mRNA, tRNA, and rRNA.

9. Use the codon chart to convert this sequence into an amino acid:

UCU-CGA-GCC-GUU-GGG-UGA

10. Explain why an mRNA strand composed of only uracil nucleotides (UUUUUUUUUU) did conclusively prove that the DNA code was a triplet code.

11. How could this mRNA sequence support a triplet code? ACACACACACAC

12. List the steps involved in protein synthesis, beginning with the DNA code in the nucleus and ending with a polypeptide. Try to explain it in 10 steps.

13. A mutation in a codon may not result in a change of an amino acid in the protein sequence. Explain why, using the genetic code chart to give an example.

Lab: RNA

 

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BIOLOGY 2.

Directions

Accurately measuring the volume of liquids, weighing chemicals, and adjusting the pH of solutions are routine procedures in a working laboratory environment. This assignment is designed to provide you with an overview of the general skills and knowledge you would need to perform such tasks.

Before completing this assignment, you should ensure you have read your textbook – particularly the section entitled pH, Buffers, Acids, and Bases. Answers should be concise and well written. Make sure you correctly explain your thought process and provide all the necessary information.

 

Question 1 

The pH of a solution describes its acidity or alkalinity: Describe how pH and H3O+ concentration are related and explain why diluting an acid raises the pH, but diluting a base lowers the pH.

 

Question 2

Phosphate Buffered Saline (PBS) is a commonly used buffer for experiments in biology because its pH and ion concentrations are similar to those in mammalian organisms. It works in a similar fashion to the blood plasma buffer mentioned in the textbook, but using dihydrogen phosphate ions and hydrogen phosphate ions for buffering through the following chemical reaction:

H2PO4- (aq)  ⇆  H+(aq) + HPO42–(aq)

 

The equilibrium arrows depict that the phosphate ion (H2PO4- ) is dissociating further into two component ions in solution, but at the same time H+ and HPO42- ions are combining simultaneously to form phosphate in solution. So, at any given point in time, and under the appropriate conditions, there is an equal quantity of dissolved ions and combined ions in solution. There is therefore always a hydrogen ion donor and an acceptor in solution.

 

Based on the equation above, which ion plays the role of hydrogen-ion donor (acid) and which ion plays the role of hydrogen-ion acceptor (base) in PBS?

 

Question 3

 

The composition of PBS is 0.137M NaCl, 0.012M Phosphate, 0.0027M KCl, pH 7.4. Below is the protocol to make 1 litre of 10x concentrate PBS.

Combine the following:

· 80g NaCl

· 2g KCl

· 14.4g Na2HPO4 (dibasic anhydrous)

· 2.4g KH2PO4 (monobasic anhydrous)

· 800mL distilled H2O

1. Adjust pH to 7.4 with HCl

2. Add H2O to 1L

3. Autoclave for 20 minutes on liquid cycle. Store at room temperature.

Which ions are being produced by this process, assuming that each of the chemical compounds dissociate into their constituent parts once they are dissolved in water?

Question 4

Preparation of the correct buffer is key to any good biological experiment and it is important that you understand how to calculate the mass of each chemical required to make that buffer and what the resulting concentration of those constituents will be in moles per litre.

Your text book explains that moles are just a way to express the amount of a substance, such that one mole is equal to 6.02 x 1023 particles of that substance. These particles can be can be atoms, molecules, ions etc, so 1 mole of water is equal to 6.02 x 1023 water molecules, or 1 mole of Na+ is equal to 6.02 x 1023  Na+ ions. Since different chemicals have different molecular weights (based on the number of protons and neutrons each atom contains) 1 mole or 6.02 x 1023 atoms of oxygen (O) will have a mass of 16g whereas 1 mole or 6.02 x 1023 atoms of sodium (Na) will have a mass of 23g

If you need more information on moles, please read Encyclopedia Britannica’s Moles website.

Although you may sometimes see it written as g/litre, the concentration of solutions is more often described in term of molarity since it better defines the chemical properties of a solution because it is proportional to the number of molecules or ions in solution, irrespective of molecular mass of its constituents. However, it is not possible to measure moles on a laboratory balance, so in the first instance chemicals are measured by mass (milligrams, grams, kilograms etc) and the number of moles is calculated using the known molecular mass (often called molecular weight and abbreviated to M.W.) of the chemical. As indicated earlier, the molecular mass of a chemical is based on the number of protons and neutrons that is contained in each atom (eg NaCl is made up of one molecule of Na, M.W. = 22.99g and one molecule of Cl, M.W. = 35.45g, so the M.W. of NaCl is 58.44g). These values can be found in the periodic table however the molecular mass of chemicals is generally provided by any vendors of the products and so can also be found on various suppliers’ websites.

 

When the concentrations of solutions are as described as ‘molar’, this refers to number of moles per litre eg a 3-molar solution of NaCl will contain 3 moles of NaCl in 1 litre of water. As indicated above, the M.W. of NaCl is 58.44g, so in 58.44g there are 6.02 x 1023 NaCl molecules ie 1 mole. So, for 3 moles of NaCl you would need to dissolve 175.32g in 1 litre of water (175.32/58.44 =3) whereas If you only dissolved 29.22g of NaCl in 1 litre of water this would result in a 0.5 molar solution (29.22/58.44= 0.5)

 

1. As directed you need to check the periodic table and pick up the atomic masses for each of the component atoms in the compounds. For example, for NaCl you need to pick the atomic weight of both sodium and chlorine and then add them to two decimal places to obtain the molecular mass of NaCl. Be sure to multiply the atomic masses by the number of individual atoms of the same element present in each compound before finally adding to the masses of other component atoms of other elements to make up the total molecular masses.

 

2. From there you can calculate the number of ‘moles’ of each compound by multiplying the provided weight of compound used in the PBS solution by their respective molar mass conversion factors (i.e. 1L divided by the molecular mass you have calculated in the first step)

 

3. Now, the molarity in Mol per Litre (mol/l) is given by the ‘number of moles’ of each compound (calculated in step 2 above) divided by the given volume of the solution.

For more information on how to calculate morality, refer to wikiHow’s 4 Ways to Calculate Molarity.

Using periodic table found in your textbook, calculate (to 2 decimal places) the molecular mass for each of the compounds used to make PBS.

Create the following table and fill it in with the mass of each component required to make 1 litre of 10 x PBS (the recipe for 10x PBS is below question 2) and their final molar concentration in the buffer calculated as described above.

 

Compound formula	Molecular mass (in g/mol)	Mass of compound per litre of 10x PBS (in g)	Molar concentration (in mol/l)
NaCl
KCl
Na2HPO4
KH2PO4

 

Question 5

As previously stated, the concentration of NaCl, KCl and Phosphate in working strength 1 x PBS is 0.137M NaCl, 0.012M Phosphate, 0.0027M KCl,  pH 7.4   How do they compare to the concentrations you calculated for 10x PBS?

 

Watch the following videos and answer the remaining questions

 “Using an Electronic Balance” from Bio-Rad tutorials

 “Using a pH Meter” from Bio-Rad tutorials

 “ Making a PBS solution ” from Community College Consortium for Bioscience Credentials

Question 6

What is the first thing to do after putting a weighing boat on the balance?

 

Question 7

If you have excess reagent on the weighing boat, what should you avoid doing and why?

 

Question 8

If you had the choice between a 1-litre beaker and a 1 litre graduated cylinder, which one should you use to measure volumes with maximal precision when making 1 litre of PBS? (you can perform an internet search to find this if you are not sure of the answer)

 

Question 9

What should be done before measuring an unknown pH of a solution using a pH meter?

 

Question 10

The recipe for PBS says to dissolve compounds in 800 ml of water, adjust the pH to 7.4, then add water up to 1 litre. The final pH should still be 7.4, because the pH of buffer solutions remains stable when they are diluted as long as the concentration of its constitutive acid and base is not too low.

 

Why do you think the protocol does not say to dissolve compounds directly in 1 litre of water?

 

Question 11

The PBS protocol above says to adjust pH to 7.4 with HCl. What does this imply on the pH of 10x PBS before adjusting the pH, would it be greater or smaller than 7.4?

 

Question 12

The last step in the protocol is to autoclave the 10x PBS solution. Why do you think this step is important? Look up the definition of autoclave if you are unsure what it means.

 

Question 13

Taking into account your response to question 5, now that you have made a 10x PBS solution, describe how you would prepare 1 litre of 1x working solution PBS, including which glassware you would use. Will you need to adjust the pH again?

 

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BIOLOGY 2

 

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