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Lab Manual - Class Index - University of Lethbridge

1. Note Flame your tubes of bacterial culture while transferring to the microfuge tube This way if there is a problem the culture can be recovered with less worry about contamination 1 Obtain 2 microfuge tubes and label accordingly Aseptically transfer 1 5 mL of culture into 38 each tube and centrifuge in a balanced configuration at 13 000 rpm for 1 minute to pellet the cells 2 Pour off the supernatant into a designated container NOT the biohazard bag the storeroom staff will hunt you down To the same tubes aseptically add another 1 mL of culture and spin as in the previous step Again pour off the supernatant Repeat until all of the 5 mL culture has been pelleted into the two microfuge tubes 2 5 mL per tube 3 Remove as much of the supernatant as possible Asepticallly transfer 1 mL of Solution toa fresh microfuge tube Use a sterile yellow micropipettor tip to add a few crumbs of lysozyme to the Solution Resuspend each pellet in 200 uL of Solution GTE by pipetting up and down Glucose Tris EDTA The Tris buffers the cells at pH 7 9 EDTA weakens the cell envelope by binding divalent cations in the lipid bilayer 4 Prepare 1 mL of Solution Il 0 2M NaOH 1 SDS Calculate the amounts of NaOH 2 M stock solution and SDS 10 stock solution Determine the amount of water required to make the volume up to 1 mL and add the water to a tube first Then add NaOH and SDS using a fresh tip for each Mix well Add 3
2. 3 Pour your gel as per instructions on page 16 Stop Point these gels will be run the morning prior to the next laboratory 26 The morning prior to the next laboratory representatives from each group will need to come in and load their samples on the gel they have prepared e For each sample 25 uL of DNA will be loaded Follow the instructions using the option involving mixing the sample on parafilm found in Laboratory 3 Remember that you will need to alter the volume of loading dye e Don t forget heat treated Lambda Hindlll e Run this gel at 70V for 1 1 5 hours e Gel may be left in ethidium bromide until lab time 27 LABORATORY 5 EXTRACTION OF DNA FROM AGAROSE SUBCLONING USING THE T VECTOR SYSTEM FROM PROMEGA Objectives To isolate purify and subclone populations of bacterial rDNA amplified from compost A EXTRACTION OF DNA FROM AGAROSE Background Reading MinElute Handbook Qiagen web site http www1 giagen com Prelab Preparations e Seta water bath to 50 C Supplies and Equipment Optima water Micropipettors Gel from Laboratory 4 MinElute Gel Extraction Kit Isopropanol Permanent marker Microfuges Transilluminator Eye protection Gloves Balance Spatulas Floating racks Waterbath Hand held UV lamp Gel tray or other work surface Procedure Work in pairs 1 Ensure that you are wearing gloves and eye protection View your gel using the hand held UV lamp using the Long Wave setting
3. 50 mM glucose 25 mM Tris Cl pH 8 0 10 mM EDTA pH 8 0 d2H20 Solution II make up just prior to using Component Stock Solution 0 2 M NaOH 2 M NaOH store in a plastic container 1 SDS 10 SDS filter sterilise Solution III KOAc Glacial Acetic Acid Component Amount for 100 mL 5 M Potassium acetate 60 mL Glacial Acetic Acid 11 5 mL d2H20 28 5 mL The resulting solution is 3 M with respect to potassium and 5 M with respect to acetate 59 Lysis Buffer 100 mM Tris HCI pH 8 0 100 mM EDTA pH 8 0 1 5 M NaCl 1 CTAB hexadecylmethylammonium bromide STET 10mM Tris HCl pH8 0 100mM NaCl 1mM EDTA 5 Triton X 100 50x TAE Tris Base 242g Glacial Acetic Acid 57 1 mL EDTA 0 5 M pH 8 0 100 mL Make up to 1L with dH20 10x TBE Tris Base 108 g EDTA 7 44 9 Boric Acid 55g Water Up to 1L 60
4. Calculate the amount of stock solution to add to your medium preparation Double check your calculations Obtain a sleeve of Petri dishes Label the agar side of each dish with a code indicating what antibiotic is present When the flask containing the medium can be held add the antibiotic flame the mouth of the flask Place back on the stir plate for 30 s to mix Pour the plates ensuring that you flame the mouth of the flask periodically Allow plates to dry for at least 24 hours at room temperature prior to using After drying place into a sleeve and store the plates at 4 C Note for plates containing tetracycline store in the dark at 4 C Questions for Discussion Why is it necessary to add an antibiotic when culturing bacteria containing a plasmid 33 e Why is it necessary to select single colonies when creating new subcultures e What is an autoclave What is the purpose of autoclaving What is a typical liquid cycle for autoclaving C TRANSFORMATION OF LIGATION MIXTURES the next laboratory Objective To transform the competent E coli DH5 cells prepared in a previous laboratory with a DNA construct containing 16S rDNA Supplies and Equipment Competent E coli DH5 cells on ice Sterilised spreaders LBAmp plates prepared in previous lab Micropipettors Waterbath set at 42 C Ice bucket ice Waterbath set at 37 C Sterile tips Racks for microfuge tubes Sterile Optima water X gal solution 2 in DNA from l
5. volume of DNA with that from another student you should have a total of 100 uL C PURIFICATION OF DNA USING THE DNEASY KIT FROM QIAGEN Prelab preparation e Ensure that a waterbath is set at 70 C Additional Supplies and Equipment Qiagen DNEasy Kit 99 ethanol 70 C waterbath Procedure Please work in pairs to clean up your DNA Notes e On page 29 of the protocol booklet under the heading Protocol for isolation of genomic DNA from crude lysates start at Step 3 DO NOT VORTEX AT ANY POINT Vortexing will shear the high molecular weight DNA After Step 4 there is no need to check the pH of the mixture proceed to Step 4 on page 17 e Elute using 100 uL of Buffer AE e Place second elution into a fresh tube do not mix elutions Label clearly Store your DNA at 20 C again ensure your tubes are very clearly labeled so that you can find them again You will quantitate your DNA either using agarose gel electrophoresis or UV spectrophotometry 21 D QUANTIFICATION OF DNA USING UV SPECTROPHOTOMETRY May or may not be performed at this point during the term Background Reading Appendix 8 Quantification of DNA Procedure 1 Prepare a 1 5 dilution of your compost DNA from the first elution Note that in order for the sample to be read you should prepare it in a final volume of 100 uL of the same solvent used to resuspend your DNA this could be water or elution buffer 2 Small groups of students will
6. Maintain Selection Pressure Plasmid vectors contain genes coding for resistance to certain antibiotics which are useful in for example transformation experiments they provide a way for a researcher to track the presence or absence of that particular piece of DNA In culturing a strain of bacteria containing a particular plasmid it is thus necessary to include an amount of the certain antibiotic to which the plasmid encodes resistance This way certain genes on the plasmid are necessary for cell survival and the plasmid is not lost when the cell divides When setting up liquid cultures of plasmid containing strains of E coli use the following chart as a guideline as to the final concentrations of antibiotic that each organism can tolerate Antibiotic Stock Conc Final Conc for mg mL E coli ug mL Ampicillin Amp 10 100 Kanamycin Km 10 50 Neomycin Nm 10 n a Streptomycin Sm 50 600 Tetracyline Tc 5 10 Gentamycin Gm 10 15 Chloramphenicol 100 Cm 10 t Dissolved in 100 ethanol May or may not be filter sterilised The rest of the antibiotics are dissolved in d2H20 and filter sterilised 49 APPENDIX 5 HANDLING OF MICROPIPETTORS Things NOT to Do Do not rotate the volume adjustor beyond the upper or lower range of the pipet Do not use the micropipettors without tips in place this could ruin the precision piston that measures the volume of fluid Do not lay down pipettor with filled tip also a
7. Y CorT 2 It is worthwhile to set up a Master Mix containing 1x PCR buffer final concentration primers water and Taq To do this calculate how many reactions you will be performing and add 1 Then calculate how much of each of the components listed are be required for all of the reactions When you are ready to set up your reactions mix all of these components well in a tube and leave on ice until needed This ensures that you don t need to pipette 0 5 uL volumes for instance 3 After preparation of Master Mix add the appropriate volume of template to each tube then add Master Mix last after all of the groups are at approximately the same stage GENTLY tap tubes to mix When everyone is ready the instructor will then show you how to operate the PCR machines If you are using the Perkin Elmer machine remember which number slot you placed your tube into that way if your label rubs off you can still locate your mixture 4 The parameters you are using for the PCR are e 5 minutes at 95 C 30 cycles of e 20 seconds at 94 C e 30 seconds at 50 C e 3 minutes at 72 C A final elongation of e 7 minutes at 72 C Questions for Discussion e What are the purposes of the primers in PCR e What happens at each temperature e How is annealing temperature determined e What is meant by stringency How can you ensure high stringency 25 e Ifyou left out the forward primer would you expect to see a band re
8. and dye if you introduce bubbles there is nothing you can do to get rid of them Draw up all of the DNA loading dye mixture ensuring that no air is left at the end of the tip bubbles in the well will cause the sample to float out Place tip into top part of well white paper underneath gel tray may help in seeing the wells and gently release digest mixture into well 11 Load remainder of DNA samples in the same way 12 Connect leads to power supply a good rule to remember is Run to Red and turn on Turn the voltage up to 70 V and run the gel for 1 5 hours The time and voltage used depends upon the type and concentration of agarose as well as on the size of DNA to be separated 13 After run is complete turn off power and disconnect leads Wearing gloves remove gel tray and gently slide gel into staining tray avoiding splashing the ethidium bromide Ethidium bromide is a mutagen and a suspected carcinogen At very dilute concentrations and with responsible handling this risk is minimised 18 14 Stain gel for 10 minutes View using transilluminator and UV light Ultraviolet light is damaging to naked eyes and exposed skin Always view through filters or safety glasses that absorb harmful wavelengths The gels will be photographed and pictures posted on the Biology 4200 web site or emailed out You will be extracting DNA from these gels and purifying it B PURIFICATION OF DNA BY EXTRACTION FROM LOW MELTING POINT AGARO
9. ingredients required Use a fresh weigh boat for each different component Rinse the spatula with distilled water and dry with a Kimwipe in between each component Rinse out the weigh boats when finished and save them Use a graduated cylinder to add the required amount of d H2O use the carboy containing Nanopure or d H20 to the flask and place the flask on a stir plate to mix the ingredients Note that unlike making a solution you do not dissolve the ingredients first in a small volume of liquid then bring up the volume For media for E coli this is not necessary although it may be necessary for more fastidious organisms Wrap foil over the top of the flask or place the top loosely onto the bottle do not tighten the lid and place a piece of autoclave tape onto the flask Autoclave the medium on liquid cycle The Following Laboratory Period 5 Media will be stored overnight in a water bath The next day complete preparation of any antibiotic solutions required Appendix 4 then remove the medium and place on a hot plate and stir gently to speed up the cooling process Preparation of antibiotic solutions Stock concentrations are provided for you in Appendix 4 Each group of 4 should prepare and filter sterilise 10 mL of the appropriate concentration of ampicillin Wear gloves when you weigh out the antibiotic The tubes should be labeled thoroughly including date of preparation concentration of antibiotic group designation
10. into the liquid in the beaker Ensure that the stir bar will NOT hit the probe e Press the READ button on the pH meter e Choose the appropriate solution to pH your solution and use a Pasteur pipette to add the solution in a dropwise fashion Allow the needle to stabilise after each drop rather than adding an entire pipetteful of solution e Once the required pH is reached press the STANDBY button on the pH meter e Lift the probe out of the solution and rinse off excess solution into the beaker using the d2H20 Be careful that you don t add too much water e Dry the probe using a Kimwipe and return to the storage solution e Transfer your solution into a graduated cylinder and bring the volume up to the required amount 53 APPENDIX 7 AGAROSE GEL ELECTROPHORESIS Agarose a linear polymer composed of alternating residues of D and L galactose joined by 1 3 and 1 4 glycosidic linkages These residues form chains of helical fibres that then aggregate into supercoiled structures with a radius of 20 30 nm When gelled a meshwork of channels with 50 200 nm diameter channels is formed Note that commercial preparations of agarose are not homogenous They may contain salts proteins or other polysaccharides for the recovery of DNA it may be necessary to use special purified grades of agarose Factors Determining Rate of Migration of DNA Through Agarose Size of DNA ds DNA migrates at a rate inversely proportional to the logi
11. melting temperature agarose can be used for recovery of DNA from the 94 agarose In terms of handling this agarose is more delicate and takes a longer time to set or gel Buffer ions are required for DNA migration Different buffers may be used We use Tris Acetate EDTA buffer TAE Although TBE has one of the higher buffering capacities of the buffers of choice much greater than Tris Acetate EDTA TAE for instance resolution of larger fragments is better in TAE DNA will move faster in TAE than in TBE and extraction of DNA from agarose may be more effective when performed on gels made with TAE Other Aspects To Consider Loading Dyes In this laboratory we make use of mixtures containing Bromphenol blue This dye migrates in 0 5x TBE at approximately the same rate as linear DNA of 300 bp in size Often xylene cyanol FF is used in conjunction with bromphenol blue or separately Xylene cyanol FF migrates at approximately the same rate as linear DNA of 4 Kb in size Markers Size standards are available as commercial preparations for instance 1Kb ladder 100 bp ladder New England Biolabs Invitrogen For an example please see http www invitrogen com content sfs manuals 15628019 pdf As commercial ladder preparations may be expensive often it is just as useful to use DNA from bacteriophage lambda that has been digested with one or two restriction endonucleases In this laboratory we generally make use of lambda di
12. our transillulminator has only one setting for UV light intensity This setting results in DNA degradation by the UV light rendering ligation less efficient Use a spatula to excise the agarose containing just the fragment of interest note this fragment should correspond to approximately 1500 bp in size Cut the band into two approximately equal fragments and place each band into a clean labeled microfuge tube Save the gel and the gels will be photographed Each pair will purify the DNA from the agarose fragment 2 Check to ensure that 99 ethanol has been added to Buffer PE before getting started Follow the guidelines on page 16 17 of the MinElute Handbook to purify your DNA Use Buffer EB for elution 28 B LIGATION OF ISOLATED DNA FRAGMENT INTO THE pGEM T VECTOR perform the same laboratory period Background Reading Promega Technical Manual for pGEM T and pGEM T Easy Vector Systems http www promega com vectors t_vectors htm or in the binder Hengen P N 1995 T vectors are plasmid cloning vectors that have been digested with EcoRV this enzyme produces blunt ends and had single 3 T overhangs added at this digest point Certain DNA polymerases such as Taq will preferentially add a single deoxyadenosine triphosphate to the 3 end of the newly synthesised DNA as reviewed in Sambrook and Russell 2001 Consequently some annealing between the T on the vector and A on the PCR product overhangs could occur Such ann
13. the University of Calgary for DNA sequencing The previous protocol is modified from the following references Birnboim H C and Doly J 1979 A rapid alkaline extraction method for screening recombinant plasmid DNA Nucleic Acid Res 7 1513 Micklos D A and Freyer G A 1990 DNA Science A First Course in Recombinant DNA Technology Cold Spring Harbor Laboratory Press Sambrook J Fritsch E F and Maniatis T 1989 Molecular Cloning A Laboratory Manual Cold Spring Harbor Laboratory Press 40 GUIDELINES FOR SAFETY PROCEDURES EMERGENCY NUMBERS City Emergency 9 911 Campus Emergency 2345 Campus Security 2603 Student Health Centre 2484 Emergency 2483 THE LABORATORY INSTRUCTOR MUST BE NOTIFIED AS SOON AS POSSIBLE AFTER THE INCIDENT IF NOT PRESENT AT THE TIME IT OCCURRED EMERGENCY EQUIPMENT Know the location of the following equipment which will be indicated to you at the beginning of the first lab 1 Closest emergency exit 2 Closest emergency telephone and emergency phone s 3 Closest fire alarm 4 Fire extinguisher and explanation of use 5 Safety showers and explanation of operation 6 Eyewash facilities and explanation of operation 7 First aid kit GENERAL SAFETY REGULATIONS 1 Eating and drinking are prohibited in the laboratory 2 Always wash your hands prior to leaving the laboratory 3 Laboratory coats are required for all laboratories 4 Report equipment problems to instructor i
14. use the UV spectrophotometer in Brent Selinger s research laboratory Please do not all pile into his lab at once 3 Turn on the spectrophotometer and choose the mode for Nucleic Acids 4 Enter the pathlength for the cell used we are using the microcells and the pathlength is 10 mm 5 Select units probably ng L 6 Do not alter the background correction option 7 Enter the dilution factor 8 Pipette 100 AL of Optima water or elution buffer into a cell Wipe off using a Kimwipe and place into the machine so that the red dot is closest to you Press the green run key This is your reference Note If you are using the larger cuvettes two sides are smooth to allow light to pass through while the other two sides are irregular resulting in readings that don t make sense 22 9 Pipette out the water Add your sample to the cell insert and press the run key Record all of the values 10 Pipette out your sample Pipette in some sterile water remove and repeat to rinse the cell out for the next pair Thought Question Evaluate your absorbance values with respect to concentration and purity 23 LABORATORY 4 THE POLYMERASE CHAIN REACTION Objective To use universal primers in the polymerase chain reaction PCR to amplify DNA from the Eubacterial domain Background Reading Same as for Laboratory 2 Atlas and Bej 1994 Prelab Preparation e Working in groups of 4 decide what reactions you need to set up note
15. 00 uL of freshly prepared Solution II to each tube Mix the contents of the tube by inversion 6 8x only Then incubate the tubes on ice for 5 minutes SDS sodium hydroxide SDS is a detergent which solublises both the lipid components of the cell membrane and the cell proteins thus lysing the cells Sodium hydroxide denatures the DNA both chromosomal and plasmid although the circular plasmid DNA strands remain intertwined Potassium acetate acetic acid Potassium acetate precipitates the SDS from the suspension taking with it the proteins and lipids which are associated with the SDS Acetic acid neutralises the pH of the solution and causes the renaturation of the plasmid DNA but only partial renaturation of the chromosomal DNA This chromosomal DNA tangle becomes trapped in the SDS mixture and as a result is precipitated out Plasmid DNA and RNA which are both smaller remain in solution 5 Neutralise the solution by adding 300 L of cold Solution Ill KOAc mix by inverting the tube and incubate on ice for 5 minutes 6 Transfer your mixture to a 2 mL tube Put on gloves and goggles move to the fume hood and extract using an equal volume of phenol chloroform 1 1 7 Remove the aqueous phase to a new tube Extract the contents with an equal volume of chloroform 8 Remove the aqueous phase to a new tube Add 2 5 volumes of 95 ethanol you may have to split up the contents into two tubes Place the tube in the freezer for 10
16. 7 OCCUPATIONAL HEALTH AND SAFETY 394 8716 EMERGENCY CALL LIST 1600 0800 Po 8A SECURITY Pp 880 5200 CHEMICAL RELEASE OFFICER 394 8937 OCCUPATIONAL HEALTH AND SAFETY 394 8716 IF THE CHEMICAL RELEASE OFFICER CANNOT BE LOCATED CALL 328 4833 DBS If the area must be evacuated all employees will be evacuated to the North Parking Lot 42 APPENDIX 1 ASEPTIC TECHNIQUE Purposes 1 To prevent the contamination of the environment and people working in the laboratory from the cultures used in the exercises 2 To prevent accidental contamination of cultures of microorganisms and of solutions and equipment used in the laboratory Correct methods of handling cultures and apparatus will be demonstrated These methods should be followed Consider carefully and remember the following points Prior to starting any work in the laboratory wash hands with soap and wash down bench area using 10 bleach This procedure should be repeated after the lab is complete Avoid working on your lab book or lab notes Clean laboratory coats must be worn If you have long hair tie it back before working in the laboratory environment Eating or drinking are not permitted in the laboratory Do not place pencils fingers or anything else in your mouth Clean air contains many bacteria and fungal spores carried on dust particles or in water droplets Any surface exposed to air quickly becomes contaminated If material is to be kept sterile it sho
17. Book Available from the course website Grading Assignments 20 Outline for Grant Proposal Due September 5 27 2007 prior to 10 00 AM 4 Laboratory Report 1 Due November 1 2006 15 prior to 10 00 AM i Grant Proposal Due November 22 2006 prior 30 to 10 00AM s Laboratory Report 2 Due December 8 2006 20 prior to 4 00 PM i Participation 10 The Participation mark will be based upon your being prepared for lab making an effort to help out not leaving the lab for extended periods of time participating in class discussions etc Failure to participate in any out of lab setup will result in a deduction of this 10 from your overall grade gt Late written work laboratory reports assignments grant proposal outlines and grant proposals must still be completed to my satisfaction and handed in within 2 weeks of the scheduled due date but will receive a grade of 0 Failure to hand in any of the aforementioned components will result in a grade of F being assigned for the course gt Late materials will not be accepted Extensions will only be considered if a written application to the Instructor including work completed on the assignment to date is made a minimum of 2 days prior to the due date gt Plagiarism will result in a grade of F in the course and a letter placed in the student s file in the Registrar s office Please consult the University of Lethbridge Calendar 2006 2007 pages 70 71 Student Expectati
18. LUTIONS When asked to prepare a solution of a certain molarity by diluting a more concentrated solution the formula to use is C1V1 C2V2 Where C1 initial concentration V1 initial volume ie the volume to be used in the preparation of the final concentration C2 final concentration the concentration of solution you are asked to prepare V2 final volume For example You have a 2M solution of NaOH and you are asked to prepare 40 mL of a 500 mM solution How do you go about this C1 2 mol L Vi unknown so let s say x C2 500 mmol L which is equivalent to 0 5 mol L V2 40 mL which is equivalent to 0 04 L Substituting these into the formula we get 2 mol L x L 0 5 mol L 0 04 L Solving for x results in x L 0 5 mol L 0 04 L 2 mol L x 0 01 L which 10 mL Remember final volume 40 mL so 10 mL of your concentrated solution of NaOH should be added to 40 mL 10 mL 30 mL of water or whatever you are asked to dilute with 48 APPENDIX 4 FINAL CONCENTRATIONS OF ANTIBIOTICS FOR CULTURING E coli Whenever you are culturing cells containing plasmids or cosmids it is necessary to maintain what is known as selection pressure ie a way of ensuring that cells continue to pass on the plasmid or cosmid DNA upon cell division Expression of plasmid encoded genes is costly to a cell so if there is no use for the genes encoded on the plasmid the plasmid may be lost when the cell divides How to
19. RATORY 2 ISOLATION OF DNA FROM COMPOST Objective To extract total DNA from a sample of compost Background Reading Amann et al 1995 Cole et al 2003 Gabor et al 2003 Pace 1997 DeLong and Pace 2001 Woese et al 1990 http pacelab colorado edu index html the Ribosomal Database Project http rdp cme msu edu index jsp The classical approach to isolation and identification of microbes in a complex environment such as soil or compost requires placing the material into a medium containing all of the necessary nutrients for growth and then incubating the medium under appropriate conditions Solid or liquid media may be used and each has particular advantages Unfortunately these techniques greatly underestimate the number of microbes present in a particular sample Atlas and Bartha 1993 For instance counts obtained using epifluoresence microscopy are usually two orders of magnitude higher than those obtained by classical culturing techniques Atlas and Bartha 1993 Other studies estimate culturability of bacteria from soil at 0 3 as cited in Roose Amsaleg et al 2001 In order to obtain more meaningful information on diversity of microbial communities in complex environments isolation and analysis of DNA from environments such as soil and compost may be performed thereby circumventing the difficulties associated with attempting to culture all microbes present Once DNA is isolated it may be studied via construction o
20. SE Prelab Preparation e Two student volunteers are needed to come in the morning prior to the laboratory in order to load and run the gels e Atthe beginning of the lab ensure a waterbath is set at 68 C Supplies and Equipment Micropipettors Waste beakers Sterile tips Optima water in kits Ethidium bromide bath UV transilluminator Gloves Spatulas Microfuge tubes Eye protection or face shields 68 C waterbath Buffer saturated phenol in the fume hood Organic waste disposal Phenol Chloroform 1 1 in the fume hood Microfuges 3 M NaOAc pH 5 2 Isopropanol 70 ethanol Goggles for working with organic solvents 95 ethanol Procedure Every student should extract his her own DNA Once the DNA is extracted from the agarose volumes will be pooled prior to Qiagen column purification Suggestion Use the 2 mL microfuge tubes initially or use the tubes labeled for phenol use only the other tubes may leak 1 Have labeled microfuge tubes handy with the lids open While wearing gloves and face 19 protection examine the gels under UV light Cut out all of the DNA visible try and avoid taking excess agarose and place the agarose plus DNA into a labeled microfuge tube If you are the last person to remove your DNA please dispose of the gel in a biohazard bag and wipe up the transilluminator 2 Melt the agarose at 68 C for 10 minutes or until you no longer see any pieces of agarose 3 Estimate the volume If it is
21. THE UNIVERSITY OF LETHBRIDGE BIOLOGY 4200 TECHNIQUES IN MOLECULAR BIOLOGY LABORATORY EXERCISES 2006 Laurie Pacarynuk Quintin Steynen TABLE OF CONTENTS Lab Exercise Page Course Outline 3 Laboratory Schedule Fall 2006 5 Laboratory 1 Solutions Preparation and Handling 7 of Microorganisms Laboratory 2 Isolation of DNA from Compost 11 Laboratory 3 Purification of Compost DNA 16 Laboratory 4 The Polymerase Chain Reaction 24 Laboratory 5 Extraction of DNA from Agarose 28 Subcloning Laboratory 6 Preparation of Competent Cells and 30 Transformation Laboratory 7 Eckhardt Gel Electrophoresis 36 Laboratory 8 Midi Preparation and Column Clean 38 up of DNA Safety guidelines 41 App 1 Aseptic Technique 43 App 2 Aseptic Preparation of Liquid Cultures 46 App 3 Dilutions 48 App 4 Final Antibiotic Concentrations for E coli 49 App 5 Handling of micropipettors 50 App 6 Use of a pH meter 53 App 7 Agarose Gel Electrophoresis 54 App 8 Quantification of DNA 57 App 9 Media and Solutions 59 Course Outline BIOLOGY 4200 5200 TECHNIQUES IN MOLECULAR BIOLOGY Fall 2006 Instructor Quintin Steynen Office D887 Phone 329 2210 email steyqj uleth ca Office Hours Monday amp Tuesday 11 00 to 12 00 or by appointment Lectures Laboratories Wednesdays and Fridays 1 00 p m 3 50 p m C740 Lab
22. alances 2x pH meters Tris NaOH pellets Nanopure H20 in large carboy Labeling tape 2x waste beakers for the pH meters Propipettors 16x bottles holding 50 mL Stirring plates Autoclave tape CTAB Gloves Pasteur pipettes bulbs 2x squeeze bottles containing d2H20 Weigh boats Scoops Disodium salt of EDTA 5M NaCl solution 5x Graduated cylinders Permanent markers Glucose 16x 100 mL beakers Stir bars Kimwipes Microbiology kits Concentrated HCI in the fume hood Safety goggles 10 mL disposable pipettes A PREPARATION OF SOLUTIONS Please see Appendix 9 for recipes General Tips Use a fresh weigh boat for each chemical Don t throw them out At the end of the lab rinse out your weigh boats and prop up so that they dry Rinse out and dry the scoops in between chemicals and reuse Use Nanopure water for making up solutions Use Optima water for resuspending DNA Prior to using the Optima water use aseptic technique hint Bunsen burners are involved to aliquot out 1 mL portions of Optima water into sterilised microfuge tubes When adjusting pH always add acid to water For autoclaving anything high pressure is involved so always leave your lids loose Procedure Note Each group should make up solutions according to Table 1 There are only supplies for each group to prepare one of each solution If you make a mistake in preparation the Instructor will supply you with solutions as you m
23. aluate your pipetting technique is the tip just filled or is a small volume of fluid left in the tube or is there an air space left in the tip of the tube While you are doing this exercise try to note what each volume should look like when in a microfuge tube Sometimes being able to eyeball a volume is a good indication if your volumes are correct and can save a great deal of problems in experiments such as PCR where errors in pipetting small volumes can result in the reaction not occurring Il LARGE VOLUME MICROPIPETTOR EXERCISE 1 You will now repeat the previous exercise using larger volumes as outlined in the following chart Tube Sol l Sol Il Sol III Sol IV 100 200 150 550 F 150 250 350 250 2 Now pool the samples as above Set your micropipettor to 1000 uL and withdraw the solution from each tube Again evaluate your micropipetting technique as above 52 APPENDIX 6 USE OF THE pH METER Prior to using the pH meter ensure that you have a supply of Pasteur pipettes Kimwipes a squeeze bottle containing d2H2O and a waste beaker e Dissolve the solute in approximately half of the final desired volume of liquid Place onto a stirring plate and stir gently e Remove the probe from the storage solution Note that the probe is glass and is VERY EXPENSIVE TO REPLACE Position the probe over a waste beaker and wash with a gentle stream of d2H20 e Carefully dry off the probe using ONLY Kimwipes NOT paper towels Place probe
24. ance pan and zero the balance e remove the first tube and place the second in the beaker Note the mass e adjust the volume in the tubes with sterile d H20 such that the balance reads the same for both tubes Place your tubes into the centrifuge in a balanced configuration ie the 2 tubes your group balanced against each other should be across from each other 3 Centrifuge the cells at 2 700 g for 10 minutes This spin will throw the cells into a pellet at the bottom of the tube 4 Remove tubes from the centrifuge Pour off the supernatant into the original flask being careful not to disturb the pellet Tap the tube gently over paper towel to remove excess supernatant and let the tube stand inverted on a piece of paper towel for 1 min 5 Add 30 mL of ice cold 80 mM MgCl 20 mM CaCl to each tube using aseptic technique Resuspend the pellet of cells by pipetting the mixture up and down Ensure that cells are completely resuspended before proceeding 6 Balance the tubes as needed and centrifuge in the same manner as above to pellet the cells Pour off the supernatant being careful not to disturb the pellet 7 Resuspend each pellet in 2 mL of ice cold 100 mM CaCl by swirling gently 31 8 Pool the contents of your tubes so that you have a final volume of 4 mL 9 Add 1 2 mL of sterile 100 glycerol and swirl gently until thoroughly mixed 10 Transfer 1004L aliquots of the competent cells into 1 5 mL microcentrifuge tubes on i
25. asmid 13 99 105 Selinger L B Personal Communication 37 LABORATORY 8 MIDI PREPARATION AND COLUMN CLEAN UP OF SUBCLONES OF INTEREST Background Reading Appendix 8 Quantification of DNA Sanger et al 1977 Hunkapiller et al 1991 GenElute handbook SIGMA Objective To prepare plasmid DNA for sequencing Prelab Preparations e The day before the laboratory each pair needs to prepare one overnight suspension culture of E coli DH5 containing the putative clone of bacterial 16s rDNA according to the instructions found in Appendix 2 Ensure that you add the correct amount of ampicillin to your tube of culture assuming a final volume of 5 mL e Prior to starting the lab ensure that a waterbath is set to 70 C Supplies and Equipment Micropipettors 5 mL of fresh overnight culture Microfuges Floating racks Permanent markers Ice bucket ice Waste beakers for bacterial waste Solution GTE ice cold Solution III KOAc ice cold 2 M NaOH 10 SDS Sterile d2H20 Isopropanol ice cold 75 ethanol Sterile tips Sterile tubes SIGMA GenElute Plasmid Miniprep kit Lysozyme 2 bottles of Phenol Chlorofom 1 1 Gloves Goggles Waste beakers labeled for phenol waste Procedure Work individually 1 tube per person When completed resuspend all pellets from 5 mL of culture in 200 L of water total then proceed to using the SIGMA kit to clean up 100 L of your DNA Save the other tube Use aseptic technique Appendix 1
26. ay suspect there is an associated participation mark penalty Table 1 Types of solutions concentrations and volumes for each group to prepare Solutions to Prepare Volumes in mL 500 mM Tris pH 8 50 500 mL EDTA pH 8 50 Lysis Buffer 50 Solution 50 STET 50 For making up the EDTA solution e After calculating the appropriate amount of EDTA required weigh out this amount and place it in a labeled beaker containing a stir bar Caution Prior to working with solid NaOH ensure that you are wearing a lab coat and gloves e add half of the amount of Nanopure water called for use the water in the carboy and place the beaker onto a stirring plate Use solid NaOH pellets to pH EDTA note that the disodium salt of EDTA will not go into solution until the pH is at 8 0 Please see Appendix 6 for guidelines on using the pH meter e Pour the solution into a graduated cylinder and adjust the volume with Nanopure water Caution Prior to working with concentrated HCI ensure that you are wearing a lab coat goggles and gloves For making up the Tris solutions e Again calculate amount required per solution add only half of the water required just enough to dissolve the compound and read the pH After adjusting to the appropriate pH stir and pour the solution into a clean graduated cylinder and adjust the volume using Nanopure water When you are finished e Ensure that solutions are labeled as
27. background readings and or appendices that the students should review and understand prior to coming to the lab Such preparation is also considered to be part of the laboratory participation grade Concept List As background preparation for the course you should ensure that you can define explain each of the following concepts gt VV VV VV VV VV VV V Gene structure what is a gene What are parts of a prokaryotic gene Transcription and translation in prokaryotes DNA replication in prokaryotes The lac operon Bacterial growth nutritional requirements techniques for isolation and cultivation of bacteria Recombination Transformation Type Il Restriction endonucleases mechanism of action formation of sticky or blunt ends Plasmid Expression vectors SDS PAGE Agarose gel electrophoresis Cloning PCR Tentative Schedule Project Sept 6 Wed Introduction and Project Overview Lab 1 Solution preparation Sept 8 Fri Lab 2 DNA isolation start Lab 3 Agarose gel preparation Grant proposals Sept 13 Wed Lab 2 DNA isolation complete Sept 15 Fri Morning Gel Electrophoresis of isolated DNA Sept 15 Fri Lab 3 Extract DNA from gel and column purify Check DNA purity and quantify start Sept 20 Wed Lab 3 Check DNA purity and quantify complete Lab 4 PCR setup Agarose gel preparation Lecture Cloning of PCR products Sept 22 Fri Lab 4 Gel Ele
28. brown in colour at this point DNA extraction protocol modified from Gabor E M deVries E J and Janssen D B 2003 Efficient recovery of environmental DNA for expression cloning by indirect extraction methods FEMS Microbiol Ecol 44 153 163 15 LABORATORY 3 PURIFICATION OF COMPOST DNA Objective To use two techniques to purify total DNA isolated from compost in order that the polymerase chain reaction may be carried out Background Reading Instructions from Qiagen DNEasy Kit Appendix 7 A PREPARATION OF LOW MELTING POINT AGAROSE GELS PLEASE REVIEW APPENDIX 7 Performed during Laboratory 2 Supplies and Equipment Low melting point agarose Scissors 2 small gel trays 2x 6 well combs Weigh boats Scoops Balance 2x 500 mL graduated cylinders 1x 100 mL graduated cylinder Microwave 2x 250 mL flasks Oven mitts Rubbermaid container for finished gels Electrophoresis tape 1x TAE Stir bars Procedure Two gels will be prepared for the entire class 1 Each gel tray holds approximately 40 mL of agarose You want to prepare a 0 5 agarose gel in 1x TAE buffer Use this information to calculate how much agarose to weigh out 2 Weigh out the appropriate amount of agarose check with the instructor if you are unsure Place into a flask and add 40 mL of 1x TAE Heat on high power in the microwave for 1 minute 16 intervals stopping frequently to ensure that the agarose mixture does not boil over 3 Swirl the flas
29. ce Cells will be frozen at 80 C to be used the next laboratory and later in the course B POURING OF LB PLATES CONTAINING AMPICILLIN to be performed during the same laboratory Objective To gain experience in preparing and pouring solid bacteriological media Note Preparation and autoclaving media will be performed out of lab Pouring media will be performed during the same lab period as preparation of competent cells Supplies and Equipment for Media Prep available in the Biology Storeroom Agar Balance Spatulas Weigh boats Tryptone Yeast Extract NaCl 250 mL Flasks Tape Permanent markers Stir bars d H20 Graduated cylinders Stir plate Aluminum foil Autoclave tape In Lab Supplies and Equipment 5x syringe filters 5x 10 mL syringes 5x sterile 15 mL Falcon tubes Ampicillin Gloves Balance 5x 50 mL beakers Scoops Weigh boats Procedure Each pair should prepare and pour 250 mL of LB Amp medium 1 Arrange a time the day prior to Laboratory 6 Part B In terms of timing weighing out the ingredients and mixing them will take about 20 minutes Autoclaving will be carried out by your instructor or the storeroom supervisor After autoclaving the medium will be stored ina water bath until the next day 2 Obtain a flask or bottle that holds 2x the volume of medium you are making Label the flask 32 5 using tape and a permanent marker and add a stir bar Use the recipe for LB found in Appendix 9 and weigh out the
30. completely as possible Include name of solution pH date and your initials or group designation Place your labeled bottles in a metal tray on the side for autoclaving At some point during the lab there will be a demo of the autoclave To prepare for this ensure that all lids to bottles are barely on the bottles you want to allow for pressure 8 changes during the autoclave cycle B HANDLING OF MICROORGANISMS Objective To practice streaking for single colonies using aseptic technique Background Reading Appendices 1 2 Additional Supplies and Equipment Liquid cultures of Escherichia coli strain Microbiology kits DH5 Racks for test tubes Tape LB plates 1 per person Inoculating loops Procedure The following should be performed using aseptic technique Appendix 1 1 Clean off your bench surface using the dettol ethanol mixture provided 2 Obtain 1 LB plate and a tube of bacterial culture 3 Following the instructions found in Appendix 2 set up a streak plate for single colonies using the broth culture of bacteria and a fresh LB plate Ensure that you label your plate completely then place your plate into the 37 C incubator in C741 Typically E coli is incubated for 16 20 hours to allow for single colony formation In the presence of antibiotics other than ampicillin incubation time may be increased up to 24 hours C PREPARATION OF AGAROSE FOR GEL ELECTROPHORESIS if time permits if not this will be p
31. ctrophoresis of PCR Products Lab 5 Extract purify PCR products from gel Sept 27 Wed Quantify purified PCR Product gel and or UV Ligation of product into a vector Grant Proposal Outline Due 10am Sept 28 Thu Lab 6 Media preparation Sept 29 Fri Lab 6 Competent cell preparation DH5 Pour media for transformation Lecture Plasmids as cloning vectors Oct 4 Wed Lab 6 Transformation of ligation products Spread plating of potential transformants Oct 5 Thu Lab 7 Replica plating for Eckhardt gel electrophoresis Oct 6 Fri Lab 7 Screen plasmids Eckhardt gel electrophoresis Lecture Expression vectors Oct 10 Tue Afternoon Lab 8 Setup liquid o n cultures Oct 11 Wed Lab 8 Alkaline lysis prep of plasmids with column clean up start Oct 13 Fri Lab 8 Alkaline lysis prep of plasmids with column clean up complete Check DNA purity and quantify gel amp spec Note DNA will be sent to U of C for sequencing Lecture Sequence analysis Project Il Oct 18 Wed Project Il Overview Primer design for rnb deletion mutations Lecture Primer design Oct 20 Fri Primer design con t Oct 25 Wed Boiling lysis plasmid prep on prnb Teasy R E digest of rnb clone and expression vector Oct 27 Fri Morning Gel Electrophoresis of digestion products 5 Oct 27 Fri Extract digestion products from agar
32. ding Why 9 Obtain tubes of DNA Set a micropipettor to 3 uL and add Loading Dye to your sample 17 Loading dye is considered to be 10x concentration you should add to your sample to create a concentration that is approximately 1x Loading Dye 1 increases density of sample ensuring that it drops evenly into well 2 adds colour to sample to simplify loading and 3 contain dyes that in an electric field move toward the anode at predictible rates Another Option In situations where you do not want to load your entire DNA sample you can cut off a small square of parafilm and dot out the loading dye in a grid pattern Remove the desired volume from your tube of DNA and mix gently on the parafilm then proceed with loading 10 Load the gel as follows Starting with the lane farthest to the right side of the gel load 7 5 uL of A DNA digested with Hindlll resuspended in loading dye Lambda DNA is at a final concentration of 0 05 pg uL Place tip into top part of well white paper underneath gel tray may help in seeing the wells and gently release digest mixture into well only pipetting to the first stop At this point decide on the order from right to left in which you will load your samples Lambda DNA digested with Hindill serves as a reference as all of the band sizes are known and can be used to compare with digested sample bands Appendix 7 Using a fresh tip for each tube pipette up and down VERY GENTLY to mix DNA
33. dsDNA and approximately 40 g mL for ssDNA and RNA For single stranded oligonucleotides an OD of 1 corresponds to approximately 33 ng mL Spectrophotometric assessment is also used to evaluate purity of nucleic acid preparations For this a ratio of the OD26 0D2 is used At 280 nm aromatic amino acids absorb strongly Absorbance values between 1 8 and 2 0 indicate that a preparation of DNA is relatively pure For RNA a value of greater than 2 0 is desireable Anything below this value may suggest contamination with guanidinium thiocyanate found in for instance the commercial solution Trizol Values lower than 1 8 indicate contamination with phenol or with proteins Readings at 230 nm close to the maximum absorbance of peptide bonds and of Tris EDTA and other buffer salts may also be performed to indicate purity of a preparation Unfortunately although spectrophotometric quantification is fast it cannot distinguish between RNA and DNA and larger concentrations at least 1 1g mL are required for reliable results Using Comparitive Intensities on an Agarose Gel If you have a very low concentration of DNA present in a solution or you suspect contamination with RNA quantitation using ethidium bromide may be more accurate In general the greater the mass of DNA present the greater the amount of ethidium bromide that will intercalate Consequently the intensity of fluorescence will reflect the mass of DNA present In the case of lambda di
34. ealing would stabilise the hybrid molecule so that ligation is more likely According to Hengen 1995 pGEM T vectors produced by Promega are the most reliable of the T vectors that are commercially available therefore this vector is the one that we will be making use of in cloning 16S rDNA amplified from compost DNA Supplies and Equipment Optima water Micropipettors Extracted DNA 5x aliquots of pGEM T vector Microfuges Permanent marker 2x Ice buckets with ice Procedure Each pair should set up one ligation e You will be setting up a ligation in a final volume of 10 uL exactly according to the instructions found on page 7 of the Promega manual e We will be leaving the ligations overnight at 4 C e One group will be setting up a ligation using vector only Background control e One group will be setting up a ligation using control insert provided Thought Questions e Diagram the reaction that is taking place catalysed by DNA ligase e What is the purpose of the Background Control If you see colonies resulting from your transformation what do they mean 29 LABORATORY 6 PREPARATION OF COMPETENT CELLS AND TRANSFORMATION A PREPARATION OF COMPETENT CELLS Objective To prepare competent E coli cells strain DH5 Transformation Background Reading Mandel and Higa 1970 Cohen et al 1972 Definition One of 3 mechanisms by which DNA is transferred from a donor cell to a recipient cell Atlas 1995 S
35. er this supernatant to the same tube used in Step 2 13 5 Repeat the extraction and incubation in Lysis buffer one additional time Repeat the centrifugation step and pool the supernatants Discard the tube containing the pellet 6 Use the gradations on the microfuge tube to estimate the volume of supernatant you have If it exceeds 1 mL split the supernatant evenly between 2 tubes Put on gloves and move to the fume hood 7 To each tube of supernatant add an approximately 50 volume of chloroform for instance if you have 500 uL of supernatant add 250 uL of chloroform Shake the tube for 1 minute Addition of an organic solvent mixing centrifuging and subsequent transfer of the aqueous mixture is often termed extraction Extraction with organic solvents such as phenol or chloroform or a mixture of the two is a standard way of removing proteins from nucleic acid solutions Deproteinisation is more efficient when two different organic solvents are used instead of one Sambrook et al 1989 Sambrook and Russell 2001 8 Place your tube in the centrifuge in a balanced configuration with tubes from other students Spin at 10000 rpm for 3 minutes 9 The aqueous phase upper layer contains your DNA Remove the supernatant to a fresh tube in the fume hood Discard the liquid chloroform waste into the container labeled organic waste and discard the tube in the beaker in the fume hood 10 To precipitate DNA add an eq
36. erformed during Day 2 of Laboratory 2 Rather than weighing out agarose and preparing a single gel each time you require one it makes more sense to create a larger volume of molten agarose let solidify then re melt and pour gels when required Each group should prepare 200 mL of 0 8 agarose in 1x TAE Additional Supplies and Equipment Agarose 250 mL bottles 1x TAE Graduated cylinders Stir bars 1 Weigh out an appropriate amount of agarose to create a 0 8 mixture check with the instructor if you are unsure ii refers to g 100 mL of solution Place agarose into a 250 mL bottle Add a stir bar 2 Use a graduated cylinder to add 200 mL of 1x TAE Heat on high power in the microwave for 1 minute intervals stopping frequently to ensure that the agarose mixture does not boil over 3 Swirl the flask each time you remove it Hold it up to the light to check to see if any agarose is not in solution you should not see any floating particles 4 Once the agarose is in solution let it cool label the bottle clearly with your group designation and store for use throughout the semester Don t remove the stir bar Thought Questions e What is the genotype of DH5 Often catalogues from suppliers of molecular biology cells and reagents have this information either in paper versions or on line e Why might E coli genotype be important in molecular biology experiments Speculate using specific examples from DH5 10 LABO
37. f BAC Bacterial Artificial Chromosome libraries for an example see Rondon et al 2000 More simply an appreciation of diversity may be obtained by using universal primers for PCR amplification of rDNA genes from the Bacterial domain on a preparation of total DNA from an environment such as compost The resulting pool of nucleotide fragments approximately 1500 bp long may then be cloned unique clones sequenced and the resulting sequences analysed in order to characterise microbes present Prelab Preparation e Ensure waterbaths are set at 37 C and 65 C Supplies and Equipment 2x vortex mixers Lysis Buffer prepared in Lab 1 Compost Balance Weigh boats Scoops Sterile 2 mL microfuge tubes Lysozyme Pronase from Streptomyces griseus Waterbath at 65 C 11 Waterbath at 37 C Floating racks 10 SDS Micropipettors and sterile tips Procedure Work individually Use 2 mL microfuge tubes 1 Weigh out 500 mg of compost avoiding any large pieces 2 Add 750 uL of lysis buffer Vortex for 5 minutes 3 Use a sterile yellow pipette tip to remove a few crumbs of lysozyme from the stock bottle Caution Lysozyme is a very fine powder and will blow off very easily Add the lysozyme to your compost mixture 4 Repeat step 3 with a fresh tip and with Pronase Cap and mix by shaking 5 Incubate your sample for 30 minutes at 37 C 6 Add 400 uL of 10 SDS to your sample Mix by vigorously shaking by hand Place samp
38. gested with Hinalll sizes of fragments and proportion of each fragment of the total genome are known Asa result comparison of intensities of unknown DNA samples in a gel with fragments of lambda may be used to provide a rough estimate of amount of DNA present To do this the unknown intensity is compared with that of the fragments of lambda For instance let s say that the unknown band of DNA 5 uL was loaded has the same intensity as that of the 9 416 Kb band of lambda Hindlll If we have loaded 0 25 yg of lambda we can perform the following calculation 9 416 Kb 0 25 ug 48 502 Kb total molecular weight of the lambda genome 0 0485 ug of unknown DNA in 5 uL 9 7 x 10 3 ug uL References 57 Sambrook J and Russell D W 2001 Molecular Cloning A Laboratory Manual Third Edition Cold Spring Harbor Laboratory Press Cold Spring Harbor Ultrospec 211 pro UV Visible Sprectrophotometer User Manual Biochrom 58 APPENDIX 9 MEDIA AND SOLUTIONS BACTERIAL MEDIA Generally make up in a flask 2x larger than volume of media Autoclave on liquid cycle then cool while stirring until flask can be held Then add antibiotics to concentrations outlined in Appendix 4 LB Broth Compound Amount for 1L 500 mL 250 mL Bacto Tryptone 10g 5g 2 59 Bacto Yeast 5 g 2 59 1 25g Extract NaCl 10g 5g 2 59 For Solid Medium 12 59 6 25 g 3 13 g Agar Solutions Alkaline Lysis Prep Solutions Solution GTE Component Final Conc
39. gested with the enzyme Hindlill Create your own map of lambda Hinalll by doing the following e Go to the web site http tools neb com REBsites index php3 e Atthe bottom of the page choose defined oligonucleotide sequences e Under Name enter Hindill e Under Oligonucleotide sequence enter AAGCTT e Scroll back to the top of the page On the right is a box containing names of standard sequences Click on Lambda e Print off the resulting picture showing fragment patterns in 0 7 agarose e Click on the enzyme name to get exact fragment sizes Print this information off also and include both pictures in your lab notebook for future reference 99 Size Determination In order to determine sizes of unknown fragments we can make use of the relationship between distance migrated from the wells and log of molecular weight By plotting log of the nucleotide pairs vs distance migrated in cm or in mm of our known for instance lambda cut with Hindlll we find that we end up with a curve that is linear for most of the relationship beyond 1 5 cm migrated Consequently by measuring the distance that the unknown migrates we can use the standard curve to determine size 56 APPENDIX 8 QUANTIFICATION OF DNA Using UV Spectrophotometry At a wavelength of 260 nm nucleic acids DNA or RNA may be quantified based on the fact that an OD optical density of 1 corresponds to a concentration of approximately 50 pg mL for
40. greater than 500 AL separate the liquid into two equal volumes in two separate tubes While wearing gloves and goggles move to the fume hood and add 0 5 volumes of buffer saturated phenol Note the phenol itself is the bottom layer of liquid in the bottle the top layer of liquid is aqueous buffer e Caution phenol is extremely corrosive 4 Mix by inversion for 1 full minute 5 Spin in a balanced configuration at 13000 rpm for 3 minutes 6 Back in the fume hood remove just the supernatant to a new tube avoid removing any of the white interface Pour out the phenol agarose mixture into the organic waste disposal in the fume hood and discard the tube and tips in the beaker provided 7 Extract one more time with phenol mixing for another minute spinning and transferring the supernatant as above 8 Extract a final time with phenol chloroform 1 1 9 Add 1 10 volume again estimate of 3M NaOAc pH 5 2 to the tube as well as 2 5 volumes of 95 ethanol Mix well and place in the freezer for 20 minutes 10 Spin your tubes for 10 minutes at 13000 rpm Discard the ethanol by pouring away from the pellet into the waste beaker 11 Wash the pellet using 500 uL of 70 ethanol flicking the tube to dislodge the pellet 20 12 Spin for 3 minutes at 13000 rpm Remove as much of the ethanol as possible as you have done in Laboratory 2 13 Let the pellet air dry for 10 minutes and resuspend in 50 uL of Optima water Pool your
41. igation dimethylformamide Gloves Microfuges Floating racks Beaker for used spreaders Procedure Work in pairs each member of the pair should be responsible for one transformation mixture 1 Obtain enough LBAmp plates for all of your transformation mixtures Do not add X gal to plates you aren t going to use today Working with one plate at a time pipette 40 uL of 2 X gal onto the surface Obtain a sterile spreader and use to spread the solution carefully and evenly over the surface of the plate Place the used spreader into the marked container for re autoclaving Repeat for the remaining plates Let these plates dry while you are carrying out the rest of the laboratory X gal or 5 bromo 4 chloro 3 indolyl 8 D galactoside is coverted by 8 galactosidase into an insoluble blue compound i X gal is prepared in dimethylformamide DMF To minimise risk wear gloves when handling the stock solution 2 Obtain 2 tubes of competent cells Thaw cells on ice as they are very fragile While thawing label 1 as ligation mix and one as control no DNA will be added to this tube For groups carrying out additional controls you will require more tubes of competent cells 34 3 To the tube labeled ligation mix add the entire contents of your ligation reaction To the control add the same volume of sterile water 4 Incubate tubes on ice for 35 minutes x It is hypothesised that this incubation allows the DNA to approach the outer cel
42. ile plate in the area shown by Diagram 1 One of 2 different methods may be followed to produce single colonies Once the first set of streaks have been made the inoculating loop is reflamed until red hot DO NOT REINTRODUCE THE LOOP INTO THE ORIGINAL CULTURE Cool the loop in the agar of the streak plate and make a second set of streaks as shown in Diagram 2 only crossing over the initial set of streaks once Flame the loop again cool in the agar and repeat for 3 more sets Diagram 3 Note try not to gouge the agar while streaking the plate 44 Diagram 1 Diagram 2 Diagram 3 45 APPENDIX 2 ASEPTIC PREPARATION OF LIQUID CULTURES OF BACTERIA CULTURE CONDITIONS FOR Escherichia coli A USING A PLATE CULTURE Obtain an agar plate containing single colonies of the desired strain of bacteria Working from single colonies ensures that the resulting culture arose from a single bacterial cell and therefore consists of a pure culture of only the organism of interest Flame an inoculating loop until red hot Lift lid of Petri dish at an angle Do not put lid down on benchtop continue to hold it until colony transfer is complete Cool loop in agar away from any colonies Pick up a colony well separated from any surrounding colonies with the edge of the loop Close lid of Petri dish Have a tube of liquid medium ready Using little finger remove cap of tube While continuing to hold lid of tube flame
43. k each time you remove it Hold it up to the light to check to see if any agarose is not in solution you should not see any floating particles 4 Once the agarose is in solution you will need to let it cool by stirring it gently so as to avoid introducing bubbles Once you can hold the flask comfortably in your hand you can pour the gel 5 While the agarose is cooling prepare the gel tray Use electrophoresis tape to cover both ends of the tray Ensure that the ends are completely sealed so that agarose does not leak out Place the comb into the slots approximately 1 cm from one end of the gel tray 6 Pour the agarose gently into the prepared tray Leave the gel at least 10 15 minutes When it doesn t move when touched transfer the whole tray to a fridge and chill Caution these gels will be very fragile 7 At the end of the lab period remove the gel from the fridge remove the tape and put the gel plus comb into a gel tray Cover the gel with 1x TAE then remove the comb The gel can remain in buffer for several days prior to using Stop Point these gels will be run the morning prior to the next laboratory Loading the gel Supplies and Equipment Power supplies Lambda DNA digested with Hindlll Loading dye Waterbath set at 65 C Parafilm Micropipettors and sterile tips Tubes of DNA Ethidium bromide bath Place tubes of DNA digested with Hindili into 65 C water bath for at least 10 minutes prior to loa
44. l surface 5 Bring your ice bucket over to the 42 C water bath Place your transformation mixtures into the water bath for exactly 1 minutes Heat shocking may allow for the formation of a temperature gradient between the inside and outside of the cell facilitating passage of DNA into the cell 6 Return your tubes immediately to the ice bucket Aseptically add 890 uL of LB to each transformation mixture Place the tubes into the 37 C water bath and incubate for 40 min This incubation allows expression of the antibiotic resistance genes It also allows the E coli to go through at least 1 generation time 7 On one plate spread 100 uL of ligation mixture competent cells Label the plate appropriately For the remaining 2 plates place both transformation mixtures the remaining DNA cell mixture plus the control lacking DNA in a balanced configuration in a microfuge and spin at 13 000 rpm for 3 minutes to pellet the cells Pour off all but approximately 100 uL of liquid from each tube into the liquid waste container Resuspend the cell pellets at the bottom of the tube in the remaining liquid and plate Ensure that your plates are clearly labeled 8 Place your plates into the 37 C incubator overnight 12 16 hours Tomorrow the instructor will remove the plates and store them at 4 C This will allow for maximum blue colour development Questions for Discussion e Why when using ampicillin as the selection agent is it necessa
45. le into the 65 C waterbath for the remainder of the lab After gel preparation and the lecture transfer your tube back into the 37 C waterbath where it will incubate overnight Also during today s laboratory Please complete Part A of Laboratory 3 Purification of Compost DNA The next laboratory period 12 Supplies and Equipment Microfuges Lysis buffer prepared in Lab 1 Waterbath at 65 C Chloroform in the fume hood Weigh boats Scoops Sterile 2 mL microfuge tubes Optima water Micropipettors Organic waste disposal in the fume hood Vortex mixer Floating racks Isopropanol Sterile tips 70 ethanol Gloves Waste beaker for tips and tubes in the 4x small beakers for liquid waste fume hood Vortex Procedure Work individually to complete extraction of compost DNA 1 Place tubes containing compost buffer mixture into the microfuge e Caution Ensure that your tubes are in a balanced configuration Get into the habit of placing your tubes with the hinges facing outward Your pellets will then always be located under the hinge a handy thing to note particularly when the pellets may not be very large 2 Spin at 8000 rpm for 10 minutes Use a pipette with a sterile tip to transfer the supernatant to a labeled sterile tube 3 Add 500 uL of Lysis buffer to the pellet Vortex vigorously for a few seconds then incubate the tube at 65 C for 10 minutes 4 Repeat the spin step outlined in Steps 1 and 2 above Transf
46. lways hold with the filled tip facing down fluid could run back into the piston Do not let the plunger snap back after withdrawing or ejecting fluid this could damage the piston Do not immerse the barrel of pipettor in fluid Do not ever flame a micropipettor tip 50 tip ejecto si Tea ii ET display To withdraw To expel To eject tip Push button to Push button to Push ejector first stop second stop Slowly release Touch tip to button side of tube yyy Figure 1 1 Use of Eppendorf Series 2100 Micropipettor Figure prepared by Katrina White Note 3 l SMALL VOLUME MICROPIPETTOR EXERCISE Each partner should try out one set of tubes Obtain 2 1 5 mL microfuge tubes Label as A and B Following the chart below add the appropriate solutions to each of the tubes Soll uL Solll uL Sollll uL SollV uL 2 3 1 4 10 5 7 3 For each solution use a fresh tip Pool and mix the reagents by placing in a microfuge in a balanced configuration Figure 6 2 Ensure that each volume is balanced with a tube containing an equal volume so balance with tubes from other groups If tubes are not balanced this will damage the microfuge motor Apply a short several second pulse to the tubes 51 Figure 6 2 Balancing samples in the microfuge rotor 4 10 uL and 25 uL were added to tubes A and B respectively Set your micropipettor s to each of these volumes and withdraw the solution from each tube Ev
47. minutes 95 Ethanol vs Isopropanol Isopropanol is more efficient an equal volume is added to a 39 solution of DNA to preferentially precipitate nucleic acids however after time it will begin to precipitate proteins 95 ethanol a larger volume 2 5x is required but precipitation is cleaner STOP POINT DNA may be stored in ethanol at 20 C until ready to continue 8 Spin the tubes in a balanced configuration in a microfuge for 10 minutes at maximum Discard the supernatant into the labeled waste beaker Wash the DNA pellet with 500 uL of 70 75 ethanol remove as much ethanol as possible then pulse in the microfuge and draw off the last few drops of ethanol Allow the tube to sit open on your lab bench for 10 minutes to let the remaining ethanol evaporate off Ethanol removes salt and any remaining SDS from the solution 9 Dissolve all DNA pellets resulting from 5 mL of culture in 200 uL of Optima H20 10 Treat 100 ul of DNA exactly like a pellet of cells and follow the protocol steps 1 through 8 found in the GenElute manual After step 2 neutralise RIGHT AWAY there is no need to wait for cells to lyse F Do you need the optional wash Why or why not Check the genotype of DH5 cells 11 Elute the DNA in 100 uL Optima water Check 5 uL of your DNA on an agarose gel DNA will also be quantified using 260 280 ratios and the four samples having the highest concentration and purity will be sent off to
48. mmediately 5 Report all spills to the instructor immediately 6 Long hair must be kept restrained to keep from being caught in equipment Bunsen burners chemicals etc SPILLS Spill of ACID BASE TOXIN Contact instructor immediately BACTERIA VIRUS SPILLS If necessary remove any contaminated clothing Prevent anyone from going near the spill Cover the spill with dilute bleach and leave for 10 minutes before wiping up DISPOSAL Upright Cardboard Boxes CLEAN LAB GLASSWEAR broken glass Pasteur pipets NO CHEMICAL BIOLOGICAL OR 41 RADIOACTIVE MATERIALS Biohazard Bags Petri plates microfuge tubes tips All of this material will be autoclaved prior to disposal BACTERIAL OR VIRAL LIQUID Tubes and flasks containing liquid cultures are placed in marked trays for autoclaving LIQUID CHEMICALS Place in labeled bottle THE UNIVERSITY OF LETHBRIDGE Policies and Procedures Occupational Health and Safety SUBJECT CHEMICAL RELEASE PROCEDURE Precaution must be taken when approaching any chemical release 1 Unknown Known Release e Clear the area e Call Security 2345 e Do not let anyone enter the area e Call Utilities at 2600 and request the air be turned off at the release site e Security will immediately notify Chemical Release Officer 331 5201 Occupational Health and Safety 394 8937 394 8716 EMERGENCY CALL LIST 0800 1600 SECURITY po 8A CHEMICAL RELEASE OFFICER ADMIN ASSISTANT 394 893
49. o of molecular weight Big particles move more slowly as they have more drag and cannot move as readily through agarose channels Concentration of agarose the higher the concentration the slower the migration Concentration of agarose will also affect separation and can be used to enhance separation of different sized fragments of DNA For instance the lower the concentration of agarose the better the separation of larger sized DNA fragments DNA conformation the three forms of DNA supercoiled form nicked circular form II and linear form Ill migrate at different rates through agarose please see the following web page for an excellent explanation and animation of the three forms and of their migration http bio classes ucsc edu bio20L animate anim2 topo htm Under the conditions we make use of it is assumed that supercoiled migrates fastest through the gel while the other two forms may not be resolveable When ethidium bromide is incorporated into agarose the ethidium bromide intercalates and will slow down stiffen the linear DNA molecules Applied voltage gels should be run at no more than 5 8 V cm note that cm refers to distance between electrodes Types of agarose Standard from 2 species of seaweed Gelidium and Gracilaria Standard agarose can be used for separation of DNA fragments from 1 25 Kb Low melting temperature modified by hydroxyethylation and consequently melts at 65 C because of this low
50. ons gt Students are expected to have completed all of the prerequisites To this end a prerequisite check is automatically performed at the beginning of the course If you have achieved less than a grade of C in the prerequisites you should be prepared to invest additional time in learning the related material Attendance at all laboratories is mandatory If you miss a laboratory it is your responsibility to make arrangements with the instructor to perform a make up laboratory that may or may not be the same as the exercise missed Note that this opportunity is dependent upon the instructor s schedule If you fail to schedule and complete a make up laboratory you will lose 5 of your final grade for each lab missed This is distinct from the laboratory participation grade Unfortunately absences of greater than 2 laboratories can not be accommodated consequently students who miss more than 2 laboratories will need to see a Student Advisor for options regarding deferring the course until the following Fall semester For certain labs students are expected to set up experiments outside of regularly scheduled lab time Effort and participation in these out of lab activities as well as in laboratory discussions of results and papers and in class participation form the basis of the laboratory participation mark Students are expected to have read the laboratories prior to coming to the labs themselves In addition most labs have associated
51. opening of tube Place end of loop containing colony into the broth and swish gently in the medium to dislodge cells Remove loop and flame top of culture tube again and replace the cap Flame the loop until red hot to kill any remaining bacterial cells Incubate 8 24 hours at 37 C for E coli 8 48 hours at 28 C for Rhizobium leguminosarum B SUBCULTURING FROM LIQUID BROTH Obtain a liquid culture of the bacteria of interest A fresh overnight culture is best but older cultures may be used Obtain the appropriate micropipettor and a sterile tip to add approximately 1 50th the amount of culture to medium and have ready Using little finger remove cap of tube While continuing to hold lid of tube flame opening of tube Tilt tube so that culture moves toward the entrance of the tube Place tip of micropipettor into culture and carefully draw up the required amount of bacteria This is done as the barrel of the micropipettor is most likely contaminated with other microorganisms so we want to avoid putting the barrel down into the test tube Flame the top of the culture tube and replace the cap 46 Remove the top of the new flask of medium and flame the top without setting down the micropipettor containing the inoculant Introduce the inoculant into the flask of fresh medium Flame the flask opening and replace the top Eject tip into the appropriate waste container Incubate 8 24 hours at 37 OC for E coli 47 APPENDIX 3 DI
52. ose gel Quantify DNA gel Nov 1 Wed Setup ligation reactions Lab Report 1 Due 10am Nov 3 Fri Competent cell prep BL21 Transform DH5 with ligation products Nov 7 Tue Afternoon Inoculate liquid cultures from transformants Nov 8 Wed Plasmid preparation Setup digests for restriction analysis Nov 10 Fri Gel electrophoresis of digests Transformation of BL21 with plasmids of interest Nov 15 Wed Morning Inoculate liquid cultures for expression testing Nov 15 Wed Small scale expression testing SDS PAGE Nov 17 Fri Setup ATW PCR using islolated plasmids Nov 22 Wed Morning Gel electrophoresis of PCR products Nov 22 Wed Agarose gel analysis Adjust PCR conditions and re run if needed Grant Proposal Due 10am Nov 24 Fri Gel purify extract PCR products if req d Setup intramolecular ligation Nov 29 Wed Transformation of BL21 with ligation mixture Dec 1 Fri Morning Inoculate liquid cultures from transformants Induction of liquid cultures Deco rien Preparation of cultures for expression testing Dec 6 Wed Small scale expression testing SDS PAGE Dec 8 Fri Lab Report 2 Due 4pm LABORATORY 1 Objective microorganisms To prepare solutions gain experience in use of the pH meter and review handling of Background Reading Appendices 1 2 3 5 and 6 Supplies and Equipment 2x b
53. pecifically transformation is the uptake of naked DNA in organisms such as E coli Bacillus and Streptococcus in a natural system this may occur when the donor cell lyses although this process is very inefficient Atlas 1995 In the laboratory to maximise this process the recipient cells must be competent In other words the cells must contain sites for binding the donor DNA at the cell surface and their plasma membranes must be in a state so that free DNA can pass across them Atlas 1995 Experiments performed by Mandel and Higa 1970 demonstrated that E coli cells could be made competent by treating them with ice cold CaCl2 and that the efficiency of this process may be improved by exposing strains of E coli to combinations of divalent cations Maniatis et a 1989 Although it is unknown by what mechanism cells become competent it is hypothesised that uptake of DNA is normally prohibited due to electrostatic repulsion between negatively charged phospholipids in the cell membrane and negatively charged DNA molecules Micklos amp Freyer 1988 Exposure to cold results in the crystalisation of the fluid cell membrane that then stabilises the distribution of the charged phosphate groups Micklos amp Freyer 1988 Cations such as Catt or Mg can then complex with exposed phosphate groups shielding the negative charges DNA molecules are then able to move through the adhesion pores in the cell membrane Micklos amp Freyer 1988 Yo
54. plate cultures Solution GTE 10 mL pipettes Propipettors Lysozyme powder 10x Loading dye Aliquots of Hindill DNA RNAse Procedure Work in groups Note each group should run an Eckhardt gel on all of the possible subclones even if the group did not obtain any Screening a large number of clones for the desired construct can often be time consuming as well as wasteful In Dr Brent Selinger s and Dr Michael Hynes laboratories they routinely use modified Eckhardt gels Eckhardt 1978 Hynes et al 1985 to examine potential Escherichia coli clones The procedure is simple and can be performed on the original transformant colonies no 36 subcultures required 1 Cast an 0 8 agarose gel for Eckhardts it is best to use 1x TBE for casting and for running the gel Use of TAE results in overheating containing 1 SDS This is accomplished with a minimal amount of bubbles by adding the 10 SDS stock solution SDS may or may not be prepared in TBE after the agarose is melted in 0 9 volumes of TBE To minimise bubbles further trickle the SDS down the side of the flask into the agarose and stir very gently to mix 2 Aseptically pipette 1 mL of Solution 1 into a microfuge tube Use a sterile yellow tip to adda scoop of lysozyme powder Add 20 uL of RNAse to the tube Close the tube and mix well Ona piece of parafilm place 15 uL droplets of Solution containing lysozyme and RNAse 3 For each colony place a Sterile tip on a sui
55. ry to remove the plates from the incubator at no later than 16 hours What are the additional colonies that develop called Do they contain the plasmid of interest Why or why not e What is blue white selection e Is blue white selection possible using any strain of E coli Why or why not e If you obtain blue colonies from your Background Control what does this mean Should you be concerned 35 LABORATORY 7 ECKHARDT GEL ELECROPHORESIS OF PUTATIVE SUBCLONES CONTAINING BACTERIAL 16s rDNA Objective To become familiar with screening colonies using Eckhardt gel electrophoresis Background Reading Eckhardt 1978 Prelab Preparation The day prior to the laboratory representatives from groups with white or pale blue colonies should replica plate these colonies onto 3 4 plates of LB Amp medium as the procedure works best with fresh cultures Each group should also pick 1 2 blue colonies to run as well When you replica plate don t just dot the colony onto the fresh medium Instead put little scribbles on the new plate so that you have little more culture to work with Is it necessary to include X gal Why or why not Incubate plates overnight 12 16 hours at 37 C Supplies and Equipment Micropipettors Parafilm Electrophoresis kits Tape 10 SDS 10x TBE stock 4x graduated cylinders 4x large beakers 4x 250 mL flasks Power supply capable of running at 5 10 volts Agarose Balance Scoops Weigh boats Microwave Fresh
56. sulting on the gel If you did explain what this would mean e Is it possible to design PCRs given only an isolatable protein Why or why not What are some of the problems associated with such an experiment How might you adapt the reaction conditions to optimise yield of desired product The protocol for PCR was modified from Whitford M F Forster R J Beard C E Gong J and Teather R M 1998 Phylogenetic analysis of rumen bacteria by comparative sequence analysis of cloned 16S rRNA genes Anaerobe 4 153 163 Also during today s laboratory Pouring an Agarose Gel Additional Supplies and Equipment Bottle of agarose 1x TAE 4 small gel trays 4x 6 well combs Oven Mitts Microwave Tape Scissors Work in groups of 4 In order to view your PCR results you will need to run the DNA out on an 0 8 agarose gel Gel trays hold approximately 40 mL of agarose One or two group members should prepare the gel tray as per Page 14 The remaining group members should take care of melting and pouring the gel 1 Loosen the top of your bottle of agarose then microwave in 1 minute bursts to liquefy the agarose Swirl the bottle each time you remove it and check the progress by holding it up to the light 2 Once all of the agarose is melted cool the bottle either by placing on a stir plate and stirring until you can comfortably handle the bottle or by swirling under running cold water and checking the temperature periodically
57. table pipettor and pick up a bit of the colony with the end of the tip Pipet the cells up and down in the droplet of solution until cells are dispersed Try not to introduce any bubbles Load the suspension into a well of the agarose SDS gels 4 Load 7 5 uL of lambda DNA digested with Hindlll as a marker 5 Once all of the samples are loaded turn power supply on to 5 10 V and run until cells are lysed i e the suspensions in the wells clear When E coli colonies are used this usually takes less than 0 5 h Following lysis increase power supply voltage to 70 V for 2 hours to resolve plasmids Upon completion of run stain gel in ethidium bromide and photograph 6 Select clones containing the plasmid of expected size Thought Questions e How many bands do you expect to see for uncut plasmid DNA What does each band represent Hint http bio classes ucsc edu bio20L animate anim2 topo htm has excellent animations and explanations e What is the expected size of a plasmid containing the desired insert How do you know e Are there any additional bands Hypothesise as to what these bands may represent References Eckhardt T 1978 A rapid method for the identification of plasmid deoxyribonucleic acid in bacteria Plasmid 1 584 588 Hynes M F R Simon and A Puhler 1985 The development of plasmid free strains of Agrobacterium tumefaciens by using incompatibility with a Rhizobium meliloti plasmid to eliminate pAtC58 Pl
58. that you need to demonstrate that what you amplify is actually bacterial DNA If you need any additional materials please let the instructor know Supplies and Equipment Micropipettors Sterile tips Sterile tubes for PCR Coloured dots PCR machines Optima water Compost DNA Microfuges 2x Ice buckets Biohazard bags Taq NEB 5x vials of 10X Buffer NEB 5x vials of 1492R 20 pmol uL 5x vials of FP1 20 pmol uL 5x vials of dNTP mix Procedure Work in groups of four Use Aseptic Technique Appendix 1 For preparation of your reaction mixtures If you are using the 250 uL tubes label on the point of the non hinged part of the lid Do not label on the sides or on the rounded part of the lid these markings will come off 1 Using the information provided in Table 4 1 calculate the final volumes of each component of your PCR mixes and fill these out in the chart handed out Decide on codes for each tube so that you know what tubes contain what mixtures 24 Table 4 1 Components and concentrations or volumes for set up of PCRs Component Final Concentration or volume Optima Water Calculated to make up the final volume to 50 uL 10x PCR buffer 1x dNTP mix 5 ul FP1 sequence 50 pmol per reaction AGAGTTYGATYCTGGCT RP1492 sequence 50 pmol per reaction TACGGYTACCTTGTTACGACT Template DNA 10 uL of each of 4 different dilutions Undiluted 1 10 1 100 1 1000 Taq DNA polymerase 0 5 uL
59. u will be preparing competent cells of E coli strain DH5a Use one of the catalogues for example GIBCO or Promega to locate the genotype of DH5 and make note of it What do all of the gene symbols mean Prelab Preparations e The day before the laboratory exercise an overnight suspension culture of E coli strain DH5 was prepared according to the directions in Appendix 2 e Approximately 2 1 2 hours before the laboratory a sub culture of mid log DH5 cells was prepared according to the directions in Appendix 2 This ensures that the culture will be in mid log phase prior to starting the procedure having an ODsso of 0 35 0 50 Supplies and Equipment 30 Ice bucket ice Racks for centrifuge tubes 100mM CaCl sterile Micropipettors 80 mM MgCl 20 mM CaCl sterile Sterile tips Glycerol sterile 1 5 mL microfuge tubes Mid log phase E coli DH5 cells 50 mL 40 mL sterile centrifuge tubes per group of 4 Balance small beaker Waste containers Sterile d2H2O for balancing beaker Procedure Note Keep cells on ice as much as possible during this procedure Perform all procedures using aseptic technique Appendix 1 and sterile equipment Work in groups of 4 to prepare cells 1 Place all solutions on ice to chill 2 Pour roughly 40 mL of mid log phase cells into 2 sterile centrifuge tubes fill the centrifuge tube to the base of the neck Balance your tubes to within 0 1 g e place 1 tube in a beaker on the bal
60. ual volume of isopropanol to each tube Mix by hand until two phases are no longer distinguishable Store the tube at room temperature for 10 minutes In solution nucleic acids are surrounded by water molecules attracted by hydrogen bonds and dipole attractions Ethanol alcohol depletes some of these molecules of water most likely by displacement Consequently the negatively charged phosphate groups on the nucleic acids are exposed When cations such as Na are added they associate with the charged phosphate groups an ionic attraction This shielding of the negative charges allows the nucleotide chains to associate closely with each other DNA associates with itself rather than with water resulting in precipitation of the nucleic acids Sambrook and Russell 2001 11 Centrifuge in a balanced configuration for 10 minutes at 13000 rpm Decant the supernatant into a waste beaker pour away from the pellet 14 12 Add 500 uL of 70 ethanol and flick the tube with your finger to dislodge the pellet Spin at 13000 rpm for 3 minutes and decant the ethanol Pulse the tube again balance your tubes put the lid on and hold down the button on the front panel of the microfuge until the microfuge is up to speed then release the button and draw off any remaining ethanol with a micropipettor 13 Leave the tube open on the bench and let the pellet air dry for 10 minutes Resuspend the pellet in 25 uL of Optima water Note that the pellet will be
61. uld be exposed only as much as is absolutely necessary for manipulation Plugs and caps of tubes tops of Petri dishes and bottles of solutions even water must not be laid on the bench nor must sterile containers and cultures be left open and exposed to the air INOCULATION OF CULTURE TUBES Again the important thing to remember is that exposure of sterile liquids or bacterial cultures to air must be minimised Ensure that you have the tubes plate of inoculum inoculating loop and a sterile tube of medium available within easy reach Flame the inoculating loop until red hot When removing inoculum from a tube remove the cap from the tube by grasping the cap between the last finger and the hand which is also holding the inoculating needle Figure 1 Do not place the cap on the bench 43 Figure 1 1 Technique for manipulating test tubes aseptically Flame the mouth of the tube by passing it rapidly through the Bunsen burner 2 3 times This sterilises the air in and immediately around the mouth of the tube Cool the loop on the inside of the tube remove the inoculum Reflame the mouth of the tube and replace the cap Flame the inoculating loop before replacing Note when removing inoculum from a plate cool the loop in the agar before picking up the bacteria STREAKING FOR SINGLE COLONIES A loop of liquid culture or a small amount of bacterial growth from a plate culture is transferred aseptically to a ster

Lab Manual - Class Index - University of Lethbridge

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