biol 2362 lab manual 2012

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BIOCHEMISTRY

BIOL 2362

FURTHER METABOLISM AND GENE EXPRESSION

LABORATORY MANUALAcademic Year (2011-2012)

Department of Life Sciences

University of the West Indies

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TABLE OF CONTENTS

Page

LAB PREPARATION & REPORT SUBMISSION 1

CITATION OF REFERENCES 6

LAB SKILLS ASSESSMENT SHEET 8

1. PRACTICAL # 1a 9

Isolation, Quantification and Purity Determination of DNA

from Rat Liver and Kidney and Cow’s Blood

2. PRACTICAL # 1b 13

Determination of the Total DNA and RNA Content

in Rat Liver, Kidney and Brain

3. PRACTICAL # 2 21

Induction and Estimation of !-Galactosidase Synthesis

In Escherichia coli

APPENDIX 1: Notes on Graphing 34

Lab Submission Data Sheets i

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LAB PREPARATION & REPORT SUBMISSION

FLOWCHART

As part of your preparation for the lab session, you are required to come to the lab with a

comprehensive flowchart outlining the procedures and your plan to undertake the lab

session in question. On the top of the page of the flowchart you should have:

! The volume of each reagent you are required to take.

! Major notes/reminders

! Expected trends/results for a given experiment.

! Questions (if any) about the procedure

Absence of your flowchart will result in an immediate deduction of marks.

IN-LAB ASSESSMENTYour lab skills and ability to produce accurate results are assessed in the in-lab

assessment. Every lab session, both you and your partner will be assessed. The final mark

will be acquired by summing the totals of all the assessment and scaling it down to an

appropriate mark which is then included in your final lab mark.

LAB REPORT SUBMISSIONBiochemistry lab reports are submitted in two parts: a Pre-lab and a Post-lab. The pre-

lab consists of the Title, Aim, Theory and Procedure and accompanying References. The

Post-lab focuses on your Results and Calculations and Discussion and must also be

referenced. Follow the instructions below strictly.

BIOCHEMISTRY PRE & POST LABS SHOULD BE TYPED (PREFERABLY) ON

LETTER SIZE PAPER OR WRITTEN ON FOLDER/BINDER PAGES. A bonus of

0.5 marks is awarded as an incentive for a well presented lab report that strictly followsthe guidelines below.

PRE-LAB SUBMISSION

NAME: LAB PARTNER:

ID# DATE (of lab session):

Title Of Lab This is given on the lab handout (e.g. Titration Curves)

Aim: This states the objective(s) of the lab, the aim of the experiment

e.g. To determine…., To investigate….

Theory: The theory should pertain to the objectives of the lab. It is a

concise introduction that gives information needed to understand

the subject and objectives of the experiment. It clearly states the

questions that the experiment sets out to answer or the hypothesis

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being tested, along with the experimental approach that was used. It

puts the experiment into perspective. YOUR INTRODUCTION

MUST BE REFERENCED. Failure to reference your work will

result in loss of marks.

Procedure: The procedure must be presented in narrative style in the past tense.

DO NOT ITEMISE OR PUT IN PUT IN POINT FORM. Include a

list of materials and reagents used at the beginning of this section.

References: See pgs 5 & 6 of this manual. FOLLOW INSTRUCTIONS

RIGOROUSLY!

POST-LAB SUBMISSION

NAME: LAB PARTNER:

ID# DATE (of lab session):

Results: The results section presents the data and observations that bear upon the

objectives of the experiment. Results are presented in tables (PLEASE DO

NOT INSERT DATA SHEETS FROM THE LAB SESSION!), figures,

graphs, calculations, diagrams, and sometimes very brief, accompanying notes.

The following is a guideline:

Tables Must:

1. Be numbered2. Have a title (this should fully describe what the table is about e.g. Table 1: Results of

Protein Calibration Curve Construction)

1. Be properly drawn (i.e. comprehensive layout of variables)

2. Incorporate all data (e.g. volume of reagents used, absorbance values, amount of

protein in mg etc.)

Calculations:

1. Only do a sample calculation (e.g. if you are constructing a protein calibration curve,

using increasing volumes of standard protein solution, you are expected to show a

calculation showing how much protein there is in a chosen aliquot of solution. Youare not expected to show the working for each aliquot that you pipette out. This is

redundant as the same method is employed.

Alternatively you may be expected to determine the protein content of a specific mass

of tissue. You may be required to do this for several different types of tissues,

however, provided that the dilution factors and processing steps are the same,

you are only required to show the working for one tissue type. Of course even if you

do not show the working, you will include all calculated values in a summary table.

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2. You must show all steps in your calculations. Marks will be lost if you do not show a

logical flow of steps.

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Graphs Must:

1. Be numbered

2. Have a title (this should be fully descriptive e.g. Graph #1: Protein calibration curve

showing how protein content (mg) varies with absorbance at 750 nm)

3. Be neatly drawn (if hand drawn use a sharp pencil point, avoid leaving stray marks

etc.)

4. Possess a scale (e.g. on horizontal axis 1 cm represents 1 unit/mg protein etc.)

3. Have labeled axes (e.g. vertical axis – Absorbance at 750 nm; horizontal axis –

Protein content in mg)

Discussion:

The discussion is a very important part of the lab script. It demonstrates a

student’s analytical skills and gives insight into how well the student understands

the lab exercise. This is where you interpret your results and compare it to

findings previously reported in literature.

The discussion should:

1. Answer the questions raised in your Aims/Objectives.

2. FOCUS ON YOUR RESULTS!!!!!

3. Show significant trends in the results

4. Point out the significance of the results/trends by comparing your findings to known

theoretical values or theories.

5. State and discuss whether the results obtained conform to the theoretical expectations

and if they do not suggest possible explanations.

6. Account for sources of error and indicate why certain precautions may have been

taken. (e.g. tissue samples kept on ice)7. Indicate why certain reagents were used (if the addition of such reagents are

important for the particular reaction).

8. BE REFERENCED to support your findings.

9. Give suggestions for improvements in experiments.

Additional Discussion:

Answer these questions separately from the discussion.

*References:

See pgs 5 & 6 of this manual. FOLLOW INSTRUCTIONS RIGOROUSLY!

FAILURE TO COMPLY WITH THE ABOVE INSTRUCTIONS WILL RESULT

IN LOSS OF MUCH NEEDED MARKS!

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LAB GRADES

Pre-labs are to be submitted on the day of the lab in question and post labs one week after

the lab date. Each part is individually marked. The mark for a given lab is produced by

summing the totals of the pre and post labs and scaling it down to 10. The marks for all

your lab sessions are then summed and this is added to the scaled-down average of your

in-lab assessments. This final mark is then scaled to 20%, which is your final lab mark

(unless otherwise stated).

Once a week after the submission date has passed, labs will not be accepted unless a very

reasonable excuse is offered.

Your lab grade accounts for 20% of your total course grade. There MAY be a practical

exam at the end of the semester. Otherwise, assessment is based on lab reports,

performance in the lab (e.g. lab manipulation skills, lab conduct, adherence to safety

regulations etc.) and any additional lab exercises (e.g. pop quizzes). Satisfactory

performance in the practicals is a prerequisite for a passing grade. Candidates who do notobtain an overall passing grade (8 out of the 20%) for the practicals will be required to

repeat the entire course.

NB PLAGIARISM IS TREATED VERY HARSHLY (whether the source of

materials is the Internet or a colleague’s report). If you are discovered plagiarizing

you will lose at least 2-3 marks out of 10 for the lab in question. Repeat offenders

will lose even more marks. The TA and demonstrators are very experienced in

detecting plagiarism, thus, it would be very unwise of you to attempt it.

COURSE ASSESSMENT

The grade awarded for biochemistry is calculated as follows:

Final examination 60%

In-course Assessment 40%

The in-course assessment comprises of two (2) written term examinations at 10% each

and performance in the practicals worth 20%.

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General Guidelines For A Good Lab Report

Layout Objectives IntroductionMaterials &

MethodsResults Discussion References Grade

!Fully labeledtables, diagramsand figures

!Within word limit

!Legible, justifiedreport

! ALL goalsclearlystated

! Concise! Based on objectives! All essential

backgroundinformation

! Contains necessarydiagrams/biochemicalreactions

! References cited in

text

! Past Tense

! No tables

! All results/analyses areclearly shown &properly tabulated

! Correct & easy tofollow samplecalculations

! Clearlydocumentedobservations/diagrams

! Concise! Observations are

well explained &reinforced byexpected orpublished results

! Includessuggestions forimprovement andimportantprecautions noted

! In-text referencescited

! Includes allcited text

! Written asindicated inmanual

Good-

Excelle

nt

! Inappropriatelylabeled Tables,Diagrams andFigures

! Exceeds wordlimit

! Script is difficultto read

! Someobjectivesmissing

! Has some mainpoints

! Missing someequations ordiagrams

! Few in textreferences

! Past Tense

! Has tables

! Incoherent layoutof results

! Difficult to follow &missing somesamplecalculations

! Incompleteobservations/diagrams

! Results arepartially explained

! Inferences are notsupported by citedreferences

! Referencecited in textbut notincluded inreference listor vice versa

Fair

! Tables,Diagrams andFigures lacktitles

! Exceeds or is

severely underword limit

! Illegibly written

! Poorlyconstructedandmissingobjectives

! Verbose! Lacks important

background points! Plagiarism detected! No diagrams/

reactions! No in-text references

! In point form! multiple

tenses! Includes

tables

! Results aremissing

! Incorrect & difficultto follow samplecalculations

! Poorly describedobservations/diagrams

! Verbose! Trends are

reported but notexplained

! Too much focus onprecautions

! No referencescited in text

! Not written asstated inmanual

! no referencelist

eak

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B. CITATION OF REFERENCES FOR THE BIOLOGICAL SCIENCES

Because all the sources of information provided in your biochemistry labs (introduction

and discussion) were not your original work, it must ALL be referenced. The

Chicago Manual of Style describes methods that are frequently used by the Biological

Sciences. We in Biochemistry will accept the author-date system of referencing.

AUTHOR –DATE SYSTEM

MAKING A CITATION WITHIN THE TEXT:

" At every point in the text at which reference is made to a particular

document, include the author's surname and the year of publication in

parentheses.

e.g. In a recent study (Smith 1982), it was shown that .....or alternatively

the reference can be found at the end of the sentence e.g. …..as previously

described (Smith 1982).

" If the author's name occurs in the sentence, give the year of publication

alone.

e.g. Smith (1982), discusses the .....

" If there are two or three authors, give the surnames of all individuals. If there

are more than three, give the surname of the first followed by et al.

e.g. Smith, Lopez and Martin (1982) reported that....

e.g. In an earlier study (Smith et al. 1982) .....

CITING REFERENCES IN A BIBLIOGRAPHY:

" Bibliography should be arranged in alphabetical order by author's surnamefollowed by year of publication.

" Use the same punctuation.

The following lists examples of citations from different sources.

1. Reference to a book:

Author(s)/editor(s). Year of publication. Title of publication in italics. Edition (if not 1st).

Publisher, place of publication. (page no. optional)

Example:

Fletcher, R. and Voke, J. 1985. Defective colour vision. Hilger, Bristol.

2. Reference to a chapter in a book:

Author(s) of chapter. Year of publication. Title of chapter in italics. In: Author(s)/editor(s)

of book. Title of book. Edition (if not 1st). Publisher, place of publication.

Example:

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Parsons, T.R. 1980. Zooplankton production. In: Barnes, R.S.K. and Mann, K.H. eds.

Fundamentals of aquatic ecosystems. Blackwell, Oxford.

3. Reference to a journal (periodical) article:

Author(s) of article. Year of publication. Title of article. Title of periodical in italics,

volume number (issue number): first page - last page. Example:

Tedder, T. and Isaacs, C. 1989. Isolation of cDNA's encoding the CD19 antigen of human

and mouse B lymphocytes. J. Immunol. 143(3): 712-717.

4. Reference to an internet source:

Author's name. Title of document, in quotation marks. Title of complete work (if

relevant), in italics or underlined. Date of publication or last revision. URL, in angle

brackets. Date of access in parentheses.

" Citing a personal site

Joseph Pellegrino, "Homepage," 12 May 1999, (12 June 1999).

" Citing a professional site

Gail Mortimer, The William Faulkner Society Home Page, 16 September 1999,

(19 November

1997).

National Association of Investors Corporation, NAIC Online, 20 September 1999,

(1 October 1999).

BIOL 2362

http://www.better-investing.org/http://www.better-investing.org/http://www.utep.edu/mortimer/faulkner/mainfaulkner.htmhttp://www.utep.edu/mortimer/faulkner/mainfaulkner.htmhttp://www.utep.edu/mortimer/faulkner/mainfaulkner.htmhttp://www.better-investing.org/http://www.better-investing.org/http://www.utep.edu/mortimer/faulkner/mainfaulkner.htmhttp://www.utep.edu/mortimer/faulkner/mainfaulkner.htmhttp://www.english.eku.edu/pellegrino/default.htmhttp://www.english.eku.edu/pellegrino/default.htmhttp://www.english.eku.edu/pellegrino/default.htmhttp://www.english.eku.edu/pellegrino/default.htm

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LAB SKILLS ASSESSMENT SHEET

Names: Date:

Cupboard #

PunctualityLate (-5)

SafetyWas not wearing gloves (-2) Improperly worn lab coat (-2)Inadequate foot ware (-2) Pours toxic waste down the sink(-5)Pours non-toxic waste into waste bottle (-3)Pools all wastes together, toxic and harmless and then disposes (-3)

Instrument Usage

Holds pipette incorrectly (-3) Leaves used tip on pipette (-2)Often slams spec cover (-3) Spills sample in spec (-4)pH meter electrode not replaced in pH buffer (-4) pH meter electrode placed on desk (-4)pH of solution taken without stirring or agitating (-4)

EfficiencyNo Flowchart (-10)Makes one or few careless mistakes (-3)Makes careless mistakes which renders entire experiment useless (-5)Has no plan, concentrations/volumes of solutions worked out in advance (-5)

Lab Etiquette & CourtesyGroup takes too much reagents (-3) Water bath more than half filled (-2)Water bath near dried out (-2) Flasks and Tubes not labeled (-2)

Accuracy & UnderstandingStudent does not understand what he/she is doing (-3)Some results are off (-1)

All results off (-3)

CleanlinessTips on desk (-3) Miscellaneous paper, parafilm on desk (-2)Pipettes on bench when not in use (-1) Left dirty utensils on desk at end of lab (-3)Places dirty utensils in cupboard (-5) Leaves workspace dirty/wet (-3)Leaves equipment on/plugged in (-3)

Breakage

BIOL 2362

Tota

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DEPARTMENT OF LIFE SCIENCES

BIOL 2362

PRACTICAL # 1a

Isolation, Quantification and Purity Determination of DNA from Rat Liver and

Kidney and Whole Cow’s Blood

Student Learning OutcomesYou should be able to:

! Extract DNA from various animal tissues! Quantify DNA using UV spectrometry! Determine purity of various DNA samples! Comment on the integrity of DNA after agarose gel electrophoresis

Isolation and quantification of DNA is an important first step in many analytical techniquesin molecular biology, genetics and biochemistry. In this lab, DNA will be extracted fromthree animal tissues using two different techniques followed by quantification and puritydetermination.

DNA isolation from animal organs involves homogenization which macerates the tissues andfrees the nuclei from the cells. Components in the isolation buffer usually dissolve themembranes, remove contaminants (mainly protein) and prevent DNA degradation. Once arelatively pure supernatant is acquired, DNA is precipitated with high salt concentrations andethanol or isopropanol. With whole blood, separation of the red blood cells must proceed first before the addition of reagents to lyse and precipitate DNA from the white blood cells.

This lab will be written up and submitted along with the following lab.

REAGENTS for Liver and Kidney DNA Isolation

Isopropanol (place in -20C freezer)

3M sodium acetate, pH 5.2 (Molecular biology grade)10% SDS1M Tris HCl, pH 8.00.5M EDTA5M NaCl

(10:1) TE, pH 7.45mL 1M Tris HCl, pH 81mL 0.5M EDTAAdd 450mL sterile distilled water, adjust pH, make up to 500mL

Proteinase K (prepare fresh)Add 5mL (10:1) TE to100mg Proteinase K. Mix to dissolve.

Lysis Buffer1mL (10:1) TE8mL 5M NaCl0.4mL 0.5M EDTAMake to 100mL with sterile distilled water.REAGENTS for Whole Blood DNA Extraction

Buffer A

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0.32M sucrose

10 mM Tris HCl

5mM MgCl2

0.75% Triton-X

Adjust pH to 7.6

Buffer B

20 mM Tris-HCl

4 mM Na2EDTA

100 mM NaCl

Adjust pH to 7.4

N.B. All solutions should be sterile. Buffer A should be autoclaved prior to addition of

Triton-X-100. Sterile filtering of solutions instead of autoclaving is a better option.

PROCEDURE for Isolation of DNA from Liver and Kidney

1. Sacrifice and remove the liver and kidneys from one rat. Weigh organs and quickly

place on ice.

2. Add 3mL/g lysate buffer to organs and homogenize. Record volume.

3. For every mL of homogenate above, add 15ul of 20mg/mL Proteinase K. Vortex.

4. Add 160uL of 10% SDS per mL of supernatant recorded in step 2. Solution may

become viscous at this point. Vortex well.

5. Place in a shaking water bath at 45C for 1 hr.

6. For every mL homogenate, add 350uL of 5M NaCl. Vortex. Record volume.

YOUR WORK STARTS HERE

7. In a clean glass test tube, take 0.6mL of each of the liver and kidney homogenate and

add 2.4mL Lysis Buffer to each. Vortex well.

8. Centrifuge both tubes at 3000 rpm for 10 minutes at room temperature.

9. Carefully pour each supernatant into individual clean, labeled test tubes. Add 0.3mL

3M sodium acetate to each tube and mix. Place on ice for 10 minutes.

10. Add 5mL of pre-cooled isopropanol from the -20C freezer. It is VERY

IMPORTANT that the isopropanol is very cold.11. Invert to mix. A visible cloudy mass should appear. This is your DNA.

12. Centrifuge at room temperature for 1 min to pillet DNA.

13. Pour off supernatant and add 3mL TE buffer (10:1). Label properly, parafilm tube and

give to your demonstrator for storage storage.

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PROCEDURE for Isolation of DNA from Whole Blood

1. Collect 40mL of cow’s blood and spin at 15000 rpm for 8 minutes.

2. Decant supernatant into the 2.5% bleach solution provided (waste) and add 5mL of

Buffer A and 10mL of cold sterile water to the pellet. Vortex gently or invert tube to

thoroughly re-suspend pellet and leave to incubate on ice for 3 minutes.

3. Centrifuge the re-suspended pellet at 5000 rpm for 15 minutes at 4oC. Discard

supernatant into 2.5% bleach solution.

4. Re-suspend pellet in 2 ml of buffer A and 6 ml of water (washing step).

5. Spin at 5000 rpm for 15 minutes at 4oC. The pellet should be white to cream in

colour. If pellet is significantly red, repeat washing step again.

6. Add 5 ml of Buffer B and 500 !l of 10% SDS to pellet. Re-suspend pellet by

vortexing vigorously for 30-60 seconds. Then add 50 !l of Proteinase K solution

(20mg/ml).

7. Leave to incubate for two hours at 55oC in a water bath. Remove samples and leave

to cool to room temperature (or leave for 2-3 minutes on ice). Add 4 ml of 5.3 M

NaCl solution. Vortex gently for 15 seconds.

8. Spin at 4500 rpm for 15-20 minutes at 4oC. Pour off supernatant into a fresh tube.

Take care not to dislodge pellet. To the supernatant, add an equal volume of cold

isopropanol (stored at -20oC). Invert 5-6 times gently to precipitate DNA.

9. Centrifuge for 1 min 12,000 rpm to pillet DNA. Wash with 1 ml of 70% ethanol.

Centrifuge as before and invert tube on tissue. Leave DNA to dry for 15-20 minutes at

room temperature. Re-suspend in 3mL of TE. Label properly, parafilm tube and give

to your demonstrator for storage.

YOUR WORK CONTINUES HERE IN THE FOLLOWING LAB SESSION

1. Centrifuge all samples at 3000rpm for 10 minutes at room temperature to remove all

traces of debris. Pour off supernatant (stock DNA) into a clean labeled test tubes.

2. Accurately pipette 250uL of your liver and kidney samples into separately labelled

tubes and make the volumes of each to 3mL with TE buffer. Do the same with 0.5mL

of your cow’s blood DNA. SAVE ALL SOLUTIONS. Some of each of your

samples will be run on an agarose gel. This will be done for you.

3. Using the appropriate cuvette, blank spectrophotometer with TE. Measure and record

the absorbance of your diluted liver, kidney and blood samples above at 260nm and

280nm.

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4. Pipette 2mL of each your liver, kidney and blood stock DNA into separately labeled

test tubes and add 1.5mL 20% (v/v) TCA solution to each. Vortex.

5. Heat all mixtures in a boiling water bath for 15 minutes. Leave to cool at room

temperature.

6. Determine the DNA and RNA concentrations of your liver, kidney and blood samples

as shown on pg 15 step 13 of the manual. DO NOT DILUTE THE EXTRACTS

HERE (lab 1 a) FOR THE RNA PROCEDURE.

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BIOL 2362

PRACTICAL # 1b

Determination of the Total DNA and RNA Content in Rat Liver, Kidney and Brain

Student Learning Outcomes

You should be able to:

! To quantify DNA and RNA via visible spectrophotometry using a destructive method.

PRINCIPLE

Many methodologies have been developed for extracting, characterizing and quantifying

DNA and RNA. Nucleic acids are often identified by their distinctive absorption spectra.

All nucleic acids absorb intensely in the ultraviolet (UV) range, a result of the conjugated

double bonds in both purines and pyrimidines. In this experiment, however, the extracted

nucleic acids will be partially degraded and converted to coloured compounds that can bemeasured spectrophotometrically in the visible range.

The extraction method used is based on the method developed by W.C. Schneider (1957)

which involves cell disruption followed by extraction with trichloroacetic acid and

ethanol. The assay procedures are based on colour reactions with the pentose moieties in

the extracted nucleic acids.

A. The estimation of DNA by the Diphenylamine reaction:

The diphenylamine reaction is specific for 2-deoxypentoses in general and is thereforespecific for DNA. The reagent contains acetic and sulphuric acids. When heated with

DNA, the acids cleave some of the phosphodiester bonds and hydrolyze the glycosidic

linkages between sugars and purines. This straight chain form of deoxypentose is

converted to the highly reactive !-hydroxylevulin aldehyde, which reacts with

diphenylamine to give a blue complex. The intensity of the blue colour, which is

measured at 600nm, is directly proportional to the concentration of DNA in the sample.

RNA does not react in the diphenylamine assay.

B. The estimation of RNA by the Orcinol reaction:

This reagent contains concentrated hydrochloric acid. When RNA is heated with acid in

the presence of ferric chloride (FeCl3.6H2O) as a catalyst, the acid cleaves some of the

phosphodiester bonds and hydrolyzes the glycosidic linkages between sugars and purines.

The hot acid also converts the ribose to furfural , which, when in the presence of ferric

ions, reacts with orcinol to produce green-coloured compounds. The orcinol reaction is

not as specific as the diphenylamine reaction as all pentoses, including the deoxyribose of

DNA, will react to some extent.

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For a given DNA and RNA solution of the same concentration, the colour intensity

produced by the orcinol reaction of DNA is one tenth that of the reaction with RNA.

Therefore the calibration curve for orcinol cannot be used to directly determine the

concentration of RNA in the TISSUE EXTRACT, as this contains a mixture of DNA and

RNA. The absorbance due to DNA is easily estimated once the concentration of DNA in

the extract has been determined. This will be 10% of the absorbance of the same

concentration of RNA on the calibration curve. Once the absorbance due to DNA is

obtained, subtract this from the absorbance reading for the extract. Using the remaining

absorbance value, determine the concentration of RNA from the calibration curve for the

orcinol reaction.

Prelab Questions:

1. What does treatment with cold TCA and hot TCA accomplish?

2. What is the purpose of adding 95% ethanol?

3. What does the RNA to DNA ratio reflect about the constituent cells?

REAGENTS

1. Diphenylamine reagent

Dissolve 1.0g of diphenylamine in 100mL of glacial acetic acid and add 2.75mL

of concentrated H2SO4, reagent quality.

2. Orcinol reagentMix together 0.2g of orcinol, 60mL conc. HCl and 0.2mL 10% (w/v) ferric

chloride solution (must be fresh).

3. Standard nucleic acid solutions

(a) DNA solution 1mg/mL

(b) RNA solution 0.2mg/mL

4. 0.1M KCl

5. 5% TCA

6. 10% TCA

7. 20% TCA

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CAUTION!

STRONG ACIDS ARE USED IN THIS PROCEDURE:

DO NOT PIPETTE BY MOUTH

USE GLASS CUVETTES

USE GLOVES AT ALL TIMES

DO NOT POUR REAGENTS INTO THE SINK. THEY WILL EXPLODE ON

CONTACT WITH WATER. POUR ALL REAGENTS INTO THE PROVIDED

WASTE BOTTLE (CONTAINING 1M NaOH), IMMERSED IN THE ICE BATH

LOCTED ON THE CENTRE BENCH.

IT IS ADVISED THAT PROTECTIVE GOGGLES SHOULD BE WORN.

IF ACID SPILLS ON YOUR SKIN, WASH IMMEDIATELY WITH SOAP AND

THEN WATER

PROCEDURE:

A. Calibration curve for DNA

1. Pipette 2mL of the stock DNA solution to a test tube and then add 1.5mL of a

20% (v/v) TCA solution.

2. Heat the mixture in a boiling water bath for 15 minutes. Leave to cool at room

temperature for 2 minutes and then quickly cool the tubes under the tap. This is

your DNA-acid hydrolysate.

3. Set up 8 test tubes as follows:

(a) Add 0.1, 0.2 (x2), 0.3, 0.5(x2) and 0.8mL of the DNA-acid hydrolysate to

labeled test tubes and then bring the final volume to 1.0mL with distilled

water.

(b) Add 2mL of the diphenylamine reagent to each tube.

(c) Prepare a blank containing 0.7mL of water, 0.3mL of 20% TCA and 2mL

of diphenylamine reagent.

(d) Mix all tubes thoroughly and boil for 10 minutes in a waterbath

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(e) Cool the tubes and measure the absorbance at 600nm after setting the

spectrophotometer at zero with the reagent blank. Plot Absorbance at

600nm against DNA content (mg).

B. Calibration curve for RNA

1. Pipette 2mL of the stock RNA solution to a test tube and add 1mL of a 20% (v/v)

TCA solution.

2. Heat the mixture in a boiling water bath for 15min. This is your RNA-acid

hydrolysate.

3. Set up 8 test tubes as follows:

(a) Add 0.1, 0.2 (x2), 0.3, 0.4(x2) and 0.5mL of the RNA-acid hydrolysate to

labeled test tubes and bring the volume to 1mL with distilled water.

(b) Add 2mL of the orcinol reagent to each tube.

(c) Prepare a blank to contain 0.7mL of 5% TCA and 0.3mL water and 2mL

of orcinol reagent.

(d) Mix all tubes thoroughly and then boil for 10 minutes in a waterbath.

(e) Cool and measure their absorbance at 660nm against the reagent blank.

Plot absorbance at 660nm against RNA content (mg).

C. Estimation of DNA and RNA in Rat Liver, Kidney and Brain Tissues

1. Kill one rat (200-250g-body weight) and remove the liver, kidney and brain and

weigh them.

2. Homogenize the tissue in 0.1M KCl (1 g tissue : 10mL of 0.1M KCl)

YOUR WORK STARTS HERE

3. Pipet 2mL of the homogenate in a glass small-rimmed test tube of approximately

12mL capacity.

4. Add 5mL of ice-cold 10% TCA to the centrifuge tube. Mix well by gentle

inversion (place a foil cap over the test tube to protect your finger).

5. Centrifuge at 1300g for 5minutes.

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6. Using a Pasteur pipette, remove and discard the supernatant. If there is a solid

layer floating on top, save this by inserting the Pasteur pipette beneath the layer

in order to aspirate the supernatant.

7. Add another 5mL of ice-cold 10% TCA to the pellet and resuspend with the

Pasteur pipette. Centrifuge and aspirate as described in steps 5 and 6 leaving two

drops of liquid above the pellet.

8. With a Pasteur pipette, disperse the pellet in the liquid above it. Then add 10mL of

95% ethanol (room temperature) to the dispersed pellet.

9. Centrifuge at 1300g for 5 min. Decant and discard the supernatant.

10. Repeat steps 8 and 9.

11. Add 5.0mL of 5% TCA (room temperature) to the centrifuge tube and disperse the

pellet with a Pasteur pipette. Place the tube in a boiling water bath for 10 minutes

agitating every few minutes.

12. Centrifuge at 1300g for 5 minutes and carefully decant the supernatant in a clean

test tube marked A (12 mL capacity). MEASURE THE VOLUME OF THE

SUPERNATANT. This nucleic acid extract will be used in the assays.

13. Determine the DNA and RNA concentrations in the acid hydrolysates as follows:

(a) In the case of DNA determination, Transfer 0.5 and 1.0mL aliquots of

the nucleic acid extract to labeled test tubes. Bring the volume to 1mL,

where necessary, with distilled water. Add 2.0mL of diphenylamine

reagent and treat as you do for the DNA calibration curve. Do the same

with your samples from lab 1a.

(b) In the case of RNA determination, dilute the nucleic acid extract 1 in 4

with water. DO NOT DILUTE SAMPLES FROM LAB 1a. Transfer

0.2mL and 0.4mL of the diluted extract to labeled test tubes. Bring the

volume to 1mL with water. Add 2mL of orcinol reagent and treat as youdo for the RNA calibration curve.

TREATMENT OF RESULTS AND WRITE UP

LAB #1a

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1. Given that an absorbance reading at 260nm of 1 (OD2601) = 50ug/mL double stranded

DNA determine the concentration of DNA in your samples on pg 3, step 3.

2. Determine the 260nm/280nm ratio for each sample (pg 3, step 3). What is the

significance of this value? Comment on your results.

3. Determine the concentration of each tissue of your DNA samples. For liver and

kidney, express this result in mg/g and blood mg/mL whole blood.

4. Of what practical application is DNA isolated from this method of use in

biochemistry and molecular biology/genetics?

5. Comment, make inferences and explain your results seen on the agarose gel.

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LAB #1a & 1b Tabulate the data for questions 2 and 3. Clearly distinguish between

the samples prepared in Lab 1a and 1b.

1. Determine the colour contribution of DNA to the orcinol reaction of the extract.

(DNA reacts one-tenth as much as the same concentration of RNA). From the

calibration curve for the orcinol reaction, find the absorbance value for an RNA

concentration that is the same concentration as the concentration of DNA in the

extract. Take one-tenth of that absorbance as the absorbance component due to

DNA and enter the values.

2. Calculate the DNA and RNA concentration (mg/g tissue) for all your samples.

3. Determine the total DNA and RNA in each organ for all your samples.

4. Do the different tissues contain different amounts of DNA and RNA? In which

tissue is the RNA concentration the highest? In which tissue is the DNA

concentration the highest? Explain the significance of your results.

5. Discuss the advantages and disadvantages of both procedures (Lab 1a and 1b) as

quantitative methods to determine DNA concentrations.

6. Compare the values acquired for liver and kidney tissues (mg/g) in Lab 1a and

Lab 1b. Explain your differences. Comment on your results.

REFERENCES:

1. Roger, L.P., Adams, John T Kowler & David P Leader. The Biochemistry of the

Nucleic Acids, 10th Edition.

2. Harper’s Biochemistry

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DATA SHEET: LAB # 1 a & b

DETERMINATION OF THE TOTAL DNA & RNA IN RAT LIVER, KIDNEY AND

BRAIN TISSUES

NAME:……………………………….LAB PARTNER:……………………………

LAB#1a

ABSORBANCE READINGS FOR DNA FROM LIVER, KIDNEY & BLOOD

Tissue Mass (g)/

600 nm 660 nm

Volume (mL)

0.5mL 1.0mL 0.5mL 1.0mL

Liver

Kidney

Blood

LAB #1b

A. CALIBRATION CURVE FOR DNA

Tube 1

(blank)

2 3 4 5 6 7 8

mL DNA-acid

hydrolysate

--- 0.1 0.2 0.2 0.3 0.5 0.5 0.8

A600nm 0.00

DNA content

(mg)

0.00

B. CALIBRATION CURVE FOR RNA

Tube 1

(blank)

2 3 4 5 6 7 8

mL RNA-acid

hydrolysate

--- 0.1 0.2 0.2 0.3 0.4 0.4 0.5

A660nm 0.00

RNA content

(mg)

0.00

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C. ESTIMATION OF DNA & RNA IN RAT LIVER, KIDNEY AND BLOOD (1a)

LIVER BRAIN KIDNEY

Mass (g)/Volume

(mL) of Tissue

----

A600nm 0.5 mL

1.0 mL

A660nm 0.2 mL

0.4 mL

D. ESTIMATION OF DNA & RNA IN RAT LIVER, KIDNEY AND BRAIN (1b)

LIVER BRAIN KIDNEY

Mass of tissue (g) ----

Final volume (mL)

of homogenate

----

A600nm 0.5 mL

1.0 mL

A660nm 0.2 mL

0.4 mL

E. DNA Results FROM LAB 1a & 1b HOMOGENATES

Concentratio of Nucleic A id (ug/g or u /mL)

Liver Kidney Blood Brain

Lab 1a Lab 1a DNA

Homogenates Lab 1a RNA

Lab 1b Lab 1b DNA

Homogenates Lab 1b RNA

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BIOL 2362

PRACTICAL # 2

Induction and Estimation of !-Galactosidase Synthesis in Escherichia coli.

Student Learning Outcomes

You should be able to:

! Prepare and assay an enzyme of bacterial origin

! Indirectly determine the level of gene expression by assaying enzyme activity

INTRODUCTION:

Enzymes synthesized by microbes can be divided into two categories:

(a) Constitutive enzymes: synthesized irrespective of the presence of the substrate forthe enzyme or a precursor of the substrate in the growth medium.

(b) Inducible enzymes: synthesized in detectable amounts only in the presence ofcompounds (termed inducers) in the growth medium, which are usually substrates (orstructurally related analogues) for the enzymes.

Note: Synthesis of inducible enzymes can often be repressed by the end product of themetabolic pathway.

Enzyme-catalyzed reactions may be followed in many ways depending on the natureof the system under study. For example, one can monitor the rate of disappearance ofa reactant or the formation of a product, the rate of gaseous exchange, the effects ofcoenzymes etc.

The activity of the enzyme !-galactosidase can be readily estimated throughcolourimetry. It is an inducible enzyme found in the bacterium, Escherichia coli,specified by the Z gene. The enzyme catalyses the hydrolysis of the disaccharide,lactose, releasing !-D-glucose and !-D-galactose. The assay method involves the useof an artificial chromogen substrate, o-nitrophenyl-!-D-galactopyranoside (ONPG),the hydrolysis of which yields o-nitrophenol (ONP). In alkaline solution, ONP has anintense yellow colour with an extinction maximum at 420 nm.

Reagents

1. LB Broth (1L)

Composition: 10g Tryptone (or peptone), 5g Yeast Extract, 10g NaCl, pH 7.4. Sterilize for 20 min in an autoclave.

2. Mineral Salts Medium (1L)

Composition: 8g K 2HPO4, 3g KH2PO4, 0.1g MgSO4.7 H2O, 1g (NH4)2SO4, 0.1% YeastExtract, 5% glucose (or lactose), pH 7.0. Sterilize glucose (or lactose) separately for 20 min.

EXPERIMENTAL

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A. Preparation of a calibration curve for estimation of o-nitrophenol.

1. Prepare a standard solution of ONP in distilled water, containing 1 µmol ONP/

mL.

2. Using this standard solution set up a series of tubes containing varying amounts of

ONP and a constant amount of alkali as outlined in the table below.

TUBE NO. ONP SOLN. (mL) Na2CO3 (mL) Distilled Water (mL)

1 0.25 1.00 3.75

2 0.50 1.00 3.50

3 0.75 1.00 3.25

4 1.00 1.00 3.00

5 2.00 1.00 2.00

6 2.50 1.00 1.50

7 0.00 1.00 4.00

3. Mix the contents of tubes thoroughly, measure absorbance at 420nm using the

contents of tube 7 as a reagent blank.

4. Plot a calibration graph relating absorbance at 420nm to ONP concentration

(µmoles/mL).

Q. 1 Is Beer’s Law obeyed over the ranges examined?

N.B. YOU ARE TO WORK IN GROUPS OF 4. Simply work with the group

directly opposite to you.

B. GALACTOSIDASE INDUCTION IN E. coli

(i) Preparation of Cell Suspensions

E. coli cells have been grown in LB Broth (25 mL) overnight. The suspension was

centrifuged and the cells resuspended in 2 mL sterile water. 1 mL was added to glucose

media (50 mL) and 1 mL to lactose media (50 mL). These were then left to grow

overnight (or approximately 18 hours). Note: these quantities quoted are for each pair of

students.

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At this point you will begin your experiment.

5. Label two centrifuge tubes glucose A and lactose A.

6. Add 10mL of the appropriate culture to the tubes. Harvest the cells by

centrifuging at 3000 rpm for 20 min. Discard supernatant.

7. Resuspend the cells in each tube using 10 mL sterile water.

8. Repeat the centrifugation and resuspension steps above.

9. Centrifuge again for a final time, discard the supernatant.

10. For the tube labeled glucose A, resuspend the pellet in 2 mL of sterile water. For

the tube labeled lactose A, re-suspend the pellet in 6 mL of sterile water.

11. From each of these tubes, take 1 mL of suspension and place into two new test

tubes labeled glucose B and lactose B respectively.

12. To the new tubes (labeled B)add 1 mL toluene. Vortex the toluene tubes for 3

min.

(ii) Estimation of !-galactosidase activity in the different preparations.

13. Pipette 4 mL of 0.1 M phosphate buffer (pH 7.2), into each of four new test tubes

labeled ‘glucose A’, ‘lactose A’, ‘glucose B’ and ‘lactose B’,

14. Add 1.5 mL 0.05M ONPG as substrate.

15. Place the test tubes in a water bath at 37 ˚C and allow the contents to attain this

temperature (wait 5min).

16. Start the reaction by adding 1 mL of the respective enzyme preparation A or B.

Note the time of the start of the reaction.

17. Shaking the tube beforehand, remove 0.5 mL samples at specific time intervals (0,5, 10, 20, 30, 40, 50, 60 min).

18. Pipette immediately into tubes containing 0.5mL 1 M sodium carbonate and

adjust the volume to 5 mL with distilled water.

19. Mix the contents thoroughly by inverting the tube five times.

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20. Clarify by centrifugation for 3 min.

21. Carefully transfer the supernatant to the cuvette and read the absorbance at 420

nm. Use 0.5 mL 1 M sodium carbonate and 4.5 mL water as your blank.

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Treatment of data

Determine the activity (units/mL) of your four stock samples.

Calculate and compare the units/ml of enzyme in each of your stock samples.

Also plot ONP concentration (umol/ml) against time (mins) and draw tangents at the

linear portions of the curves to determine U/ml (initial velocity) for each sample.

Discuss your results fully.

REFERENCES:

1. Lederberg, J. (1950). J. Bact. 60. 381.

2. Hestrin, S., Feingold, D. S., & Schramm, M. Methods in Enzymology. Vol. I. Ed.

P. Colowick & N. O. Kaplan. p. 241.

3. Kaempfer, R.O.P. and Magasanik, B. (1967). J. Molec. Biol. 27. 475.

4. Robert F. Boyd (1988) General Microbiology. 2nd Ed. Times Mirror, Mosby

College Publishing p. 237-238.

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BIOL 2362

DATA SHEET: LAB # 2

INDUCTION AND ESTIMATION OF !-GALACTOSIDASE IN E.coli

NAME:………………………………..LAB. PARTNER:………………………………..

A. Calibration curve for the estimation of o-Nitrophenol

TUBE [ONP] µmoles/mL A420nm

1

23

4

5

6

7*

*reagent blank

B. Galactosidase Induction in E.coli

Absorbanc at 420nm

Time (min.) Glucose A Glucose B Lactose A Lactose B

0

5

10

20

30

40

50

60

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APPENDIX 1: NOTES ON GRAPHING

A. DRAWING GRAPHS BY HAND

A graph is a plot of two quantities. One is something that you control and is called the

independent variable. Usually it goes on the x-axis. The other quantity that changes as

you change the independent variable is called the dependent variable. This goes on the

y-axis.

For example if you put varying volumes of a protein solution in a series of test tubes andthen measure the absorbance, the variable you control is the quantity of protein, so it goes

on the x-axis. The absorbance is what changes as you change the protein quantity, so it

goes on the y-axis.

Always use graph paper when drawing graphs.

UNITS AND EXPONENTS

Assume you did a protein determination and recorded the following data:

Protein Content (mg) Absorbance reading at X nm

0.001 0.05

0.003 0.16

0.005 0.225

0.007 0.33

0.008 0.375

The best thing to do is to change the units to easier numbers to be plotted. For example,

multiply the values by 1000 so that you obtain readable values like 1, 3, 5, 7 and 8. Then

on your graph you can use exponents to reflect this. The x-axis would now be labelledmg x 1000 or mg x 103. Additionally if your values are very large numerically, one can

simply find the log10 value and plot points using a log scale. Remember to take the

antilog for any value obtained from the graph that will be used in further calculations .

VARIABLE RANGE In order to determine an appropriate variable range; subtract the lowest data value from

the highest data value. Do each variable separately.

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Figure 1 . (a) scatter plot (b) line of best fit. NB that the best-fit line does not necessarily pass

through any of the points plotted.

BAD DATA POINTS

You are the one to decide when you have a bad data point. The computer does not make thisdecision for you. Table 1 shows a data series that has been plotted on the graph shown inFigure 2. Notice that 5 of the 6 points fall perfectly on a straight line. However one point (30,0.05) is way off. Your first action should be to redo the 30-µg tube rather than report data ofthat calibre. If that is not possible, simply draw the line through the 5 “perfect” points andignore the bad point.

Table 1: Absorba ce vs. mg Protein

mg protein Absorbance

0 0.00

10 0.10

20 0.20

30 0.05

40 0.40

50 0.50

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0

0.1

0.3

0.4

0.5

0 13 25 38 50

A b s o r b a n c e @ 7

5 0 n m

Protein content (ug)

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Figure 2. Graph with a bad data point

USE THE WHOLE GRAPH

It is best to use as much of the graph as possible. Also, the closer the line is to a 45˚

angle, the more accurate you will be when you interpolate an unknown from it. DO NOT

extrapolate the line beyond the last data point especially in the case of calibration

curves. Refer to Beer-Lambert’s Law and the section on Spectrophotometry.

TITLE:

Your graph must be numbered and have a descriptive title. For example, Graph #1:

Protein Calibration Curve.

B. GRAPHING WITH COMPUTERS

There are many graphing programs available but Excel is the most popular choice at this

level. Therefore we will use Excel to demonstrate how you can analyse data using

spreadsheets and graphing programs.

Loading the Spreadsheet

The first step is to set up the spreadsheet. With Excel you have a basic grid of columns,

which are labelled A-Z, and rows, which are numbered one to several hundred. Each

combination of a number and letter is called a cell. You start by putting data into the cells.Cells can hold data in the form of numbers or letters. If you wanted to plot absorbance vs.

mg of protein for the Lowry assay, you might have data that looks like Table 2.

When you load this into Excel, you could put the column labels (mg Protein and

Absorbance into the spreadsheet. The program will talk you through the creation of the

graph, including labelling the axes. It is best to put the values that will be on the x-axis

into the left-hand column, as it simplifies the graphing process. Figure 3 shows what the

spreadsheet would look like.

Table 2: Absorbance versus mg Protein for the Lowry Assay

mg Protein Absorbance

0 0.00

10 0.10

20 0.21

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30 0.29

40 0.40

50 0.52

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Figure 3. Excel spreadsheet.

Creating the Graph

Once you have loaded the data into the spreadsheet, you are ready to create the graph.First you need to select the data that will be put into the graph, so click on the A1 cell and

drag down and over until all of your data is highlighted. Then you click on the wizard

button. The first dialog box gives you a choice of chart types, such as bar graph, pie

graph, scatter plot and so on. Normally you will want to do the XY scatter plot, so this

will be used as the example.

There is a potential danger in using the computer to make the graph! The default value

for many programs would yield straight lines connecting the dots. THIS IS WRONG IN

BIOCHEMISTRY!

You want the best fit line of some type. In your case it would be a linear regression line.Choose the option XY scatter that does not attempt to put any line over the points. The

lines will be put in later. Click on “Next” to continue the dialog. The next box (box 2 of

4) asks about chart source data. One you have correctly selected the data from your

column, you can go on to the following box by clicking on the “Next”

icon. This brings you to box 3 of 4 in the dialog process. This is the chart-formatting step

where you decide how you want the graph to look. There is a “title”: tab you can select

and then add in the graph title and the title of both axes. There is a “gridline” tab that

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determines the number of visible gridlines. The fourth dialog box gives you the option of

putting the graph as an object on the spreadsheet itself or making it a separate page.

SELECT THE LATTER.

Remember to always select for a graph that goes through the origin when plotting

calibration curves.

To avoid creating the default “connect the dots” graph, we selected a graph that had no

line. You therefore still need to connect the line. Create the line using the pulldown

menus on the top bar. Choose “chart” and then “ add trendline” choice from the pulldown

menu. The box that pops up gives you a picture of possible types of lines, including linear

regression and polynomial. Select “linear regression” for linear data.

Non-Linear graphs

Such graphs would start off the same way but you would use the chart wizard differently

to customise your graph. A common example would be when analysing enzyme kinetics

data.

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BIOL 2362

DATA SUBMISSION SHEET: LAB # 1a & b

DETERMINATION OF THE TOTAL DNA & RNA IN RAT LIVER, KIDNEY AND

BRAIN TISSUES

NAME:……………………………….LAB PARTNER:……………………………

LAB#1a

ABSORBANCE READINGS FOR DNA FROM LIVER, KIDNEY & BLOOD

Mass (g)/

600 nm 660 nm

Volume (mL)

0.5mL 1.0mL 0.5mL 1.0mL

Liver

Kidney

Blood

LAB #1b

F. CALIBRATION CURVE FOR DNA

Tube 1

(blank)

2 3 4 5 6 7 8

mL DNA-acid

hydrolysate

--- 0.1 0.2 0.2 0.3 0.5 0.5 0.8

A600nm 0.00

DNA content

(mg)

0.00

G. CALIBRATION CURVE FOR RNA

Tube 1

(blank)

2 3 4 5 6 7 8

mL RNA-acid

hydrolysate

--- 0.1 0.2 0.2 0.3 0.4 0.4 0.5

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A660nm 0.00

RNA content

(mg)

0.00

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H. ESTIMATION OF DNA & RNA IN RAT LIVER, KIDNEY AND BRAIN TISSUES

LIVER BRAIN KIDNEY


Final volume (mL)

of homogenate

----

A600nm 0.5 mL

1.0 mL

A660nm 0.2 mL

0.4 mL

I. ESTIMATION OF DNA & RNA IN RAT LIVER, KIDNEY AND BRAIN (1b)

LIVER BRAIN KIDNEY


Final volume (mL)

of homogenate

----

A600nm 0.5 mL

1.0 mL

A660nm 0.2 mL

0.4 mL

J. DNA Results FROM LAB 1a & 1b HOMOGENATES

Concentratio of Nucleic A id (ug/g or u /mL)

Liver Kidney Blood Brain

Lab 1a Lab 1a DNA

Homogenates Lab 1a RNA

Lab 1b Lab 1b DNA

Homogenates Lab 1b RNA

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DATA SUBMISSION SHEET: LAB # 2

INDUCTION AND ESTIMATION OF !-GALACTOSIDASE IN E.coli

NAME:………………………………..LAB. PARTNER:………………………………..

C. Calibration curve for the estimation of o-Nitrophenol

TUBE [ONP] µmoles/mL A420nm

1

23

4

5

6

7*

*reagent blank

D. Galactosidase Induction in E.coli

Absorbanc at 420nm

Time (min.) Glucose A Glucose B Lactose A Lactose B

0

5

10

20

30

40

50

60

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