Precut Lambda Dna

Precut Lambda Student Instructions and Checklist (updated 9-28-10)

This document includes detailed student instructions for the Precut Lambda lab in a checklist format. Included are instructions on:  how to make agarose gels  how to store gels  how to place the gels into the electrophoresis chamber  how to use the mini centrifuge  how to use micropipets  how to stain and destain gels

IMPORTANT! Safety of students is the teacher’s responsibility. Prior to using this instruction checklist, the power supply, and the electrophoresis chamber with students, the teacher should be familiar with the:  the Biotechnology Explorer Analysis of Precut Lambda DNA Kit Instruction Manual given to teachers at the SEEDBEd and MEDBEd Workshops for High School Teachers,  the Bio-Rad safety precautions for the Bio-Rad PowerPac Basic Power Supply found at http://www.bio-rad.com/cmc_upload/Literature/51585/4006213E.pdf  the Bio-Rad safety precautions for the Bio-Rad Wide Mini-Sub Cell GT Electrophoresis Chamber found at http://www.bio-rad.com/cmc_upload/Literature/38717/M1704400B.PDF

1 Captain______PRECUT LAMBDA DNA LAB INSTRUCTIONS...CHECKLIST FOR STUDENTS PART “A”: MAKING THE AGAROSE GEL

_____ Please skip this page and the next page if the gels have already been prepared for you.

_____ Wear gloves, goggles, & lab coat/apron.

_____ Choose team captain to keep team focused on following the instructions on this checklist. At the top of this page, the captain will list the names of all team members.

_____ If a Workstation Inventory form is available, designate one person in your team to confirm that all of the listed supplies are in your work area now and at the end of lab. Notify instructor if something is missing.

_____ Clean the chemical scoop with a paper towel dampened with 70% alcohol.

_____ Secure 1 piece of weighing paper or 1 weighing boat, the agarose powder, and access to a balance.

_____ Place weighing paper/boat on the balance pan and “zero the balance.” With the clean dry scoop, measure .70 grams of agarose powder onto the weighing paper. ( .7 grams of agarose powder is about ¼ level teaspoon)

_____ Pour the agarose powder from the weighing paper into a 125 ml Erlenmeyer flask

_____ Measure 70 ml of 1x TAE solution in a 100 ml graduated cylinder. Place the cylinder on a level surface and read the bottom of the meniscus at eye level.

_____ Add the 70 ml of TAE to your agarose powder in the flask.

_____ Swirl the liquid in the flask for about 10 seconds

_____ Secure hot pads and access to a microwave oven

_____ Heat the agarose/TAE solution for 30 seconds in the microwave oven.

_____ Using a hot pad, remove the solution from oven and swirl for about 10 seconds.

_____ Again, heat the solution for 30 seconds in the microwave

_____ Again, using hot pad, remove the flask from oven and swirl for 10 seconds

_____ Check to see if the agarose powder is all dissolved and gel solution is transparent. If the solution is not clear, heat the solution for 15 seconds, swirl and recheck. Repeat this step if necessary. Please note that the solution should momentarily reach a slow boil and then be immediately removed from the microwave oven. Avoid rapid boiling because steam will escape quickly and the agrarose solution will become too concentrated.

_____ When the solution is clear, allow it to cool at room temperature for approximately 12 minutes to about 60˚C. Record the starting time of the cooling period. While you are waiting for the solution to cool, continue to follow the steps on this checklist.

2 _____ Rinse the scoop with water at the sink. Clean the scoop with a paper towel dampened with alcohol. Dry it and put it away. Wash, dry, and put away graduated cylinder.

_____ Secure a leveling bubble, gel tray, gel comb, two dams and one electrophoresis chamber. With a paper towel dampened with 70% alcohol solution, wipe the surfaces of these items.

_____ Insert the gel tray and dams into the electrophoresis chamber as shown by your instructor. Do not add the solution yet.

_____ Place the leveling bubble in the tray. If the air bubble is not centered in the window inform your instructor. A level tray will produce a gel with uniform thickness.

_____ Fit the gel comb handle into the two gel tray slots that are about 1 cm from the dam nearest the black electrode end of the chamber.

_____ IMPORTANT!! Ask your instructor to check your gel tray installation, the leveling bubble, the comb installation, and the temperature of your gel solution before proceeding to the next step. (If your solution has cooled too much and has turned into a cloudy gel, you will need to reheat the gel until it becomes a clear liquid again. Then allow the reheated solution to cool to about 60°C.)

_____ Return the leveling bubble to its proper storage location.

_____ After securing instructor approval, pour the gel solution into the gel tray. With a clean pipet tip, “pop” any bubbles or “pull” them to the edges of the gel. Record the starting time and allow to cool for about 30 minutes or until firm. Do not move the solution while it is cooling. The solution will become cloudy as it becomes a gel.

_____ While the solution is turning into a gel, wash the flask and put it away.

_____ When the gel is firm and has cooled to room temperature, remove the comb from the gel by slowly rocking the comb and pulling upward slowly. Clean the comb and return it to the storage area.

_____ Carefully remove the dams (casting gates) from the chamber so as not to tear the gel. Rinse and dry the dams and return them to the storage area. Skip the next 3 steps if you will be using the agarose gel immediately.

_____ If you wish to store the gel in the gel tray and in the electrophoresis chamber until the next day, completely fill the reservoirs and cover the gel with TAE solution to keep the gel from drying out. This may sit at room temperature overnight. Skip the next 2 steps.

_____ If you wish to store the gel, overnight, in the gel tray but removed from the electrophoresis chamber, secure a resealable gallon size plastic bag and a permanent marker. Label the plastic bag with your name and date. Add about 50 ml of TAE to the bag to keep the gel moist. Store tray with gel and TAE in the bag at room temperature or in the refrigerator. Skip the next step.

_____ If you wish to store the gel for 2 to 7 days, secure a resealable quart size or gallon size plastic bag and a permanent marker. Label the plastic bag with your name and date. Slowly and carefully slide the gel out of your gel tray into your plastic bag. CAUTION: the fragile gel will break and tear if you are not careful. Add about 50ml of TAE solution to the bag to keep the gel moist. Store in the refrigerator. Rinse the gel tray with water and clean it with a paper towel dampened with 70% alcohol solution. Return tray to the storage area.

_____ If you are working with a Workstation Inventory form, check it now and notify your instructor if something is missing. If you are finished with this lab for today, give your Workstation Inventory to your instructor at this time.

3 Captain:______PRECUT LAMBDA DNA LAB INSTRUCTIONS...CHECKLIST FOR STUDENTS PART “B”: LOADING THE GEL, ELECTROPHORESIS, STAINING, & DRYING

_____ Wear goggles, gloves, and lab coat/apron.

_____ Choose team captain to keep team focused on following the instructions on this checklist. At the top of this page, the captain will list the names of all team members.

_____ If a Workstation Inventory form is available, designate one person in your team to confirm that all supplies are in your work area now and at the end of lab. Notify instructor if something is missing.

_____ IMPORTANT: If your gel was stored without the gel tray, carefully and slowly return the gel to the tray. The fragile gel can easily tear without adequate support. The well openings need to face upward.

_____ Place the gel tray into the electrophoresis chamber as shown by your instructor. The wells made by the comb should be nearest to the black (cathode) negatively charged end of the chamber. The DNA will migrate toward the red (anode) positively charged end of the chamber during electrophoresis because the phosphate (PO4) in the DNA has a negative charge. (Recall that opposite charges attract each other.)

_____ IMPORTANT: Ask your instructor to check the position of your gel in the electrophoresis chamber and to initial this step before you proceed to the next step.

_____ Pour enough TAE solution into the gel tray and reservoirs to completely cover the gel with approximately 2 mm of solution. You will need approximately 275ml of TAE.

_____ Review the following procedure for using micropipets: a. Do not lay the micropipet on the table. When not in use return it to the micropipet stand. b. Adjust the volume dial if needed. c. Attach a clean pipet tip to your micropipet. d. In a sitting position, with both elbows on the table, hold the micropipet in a vertical position while in use. e. Press the micropipet plunger to the FIRST stop. f. Insert the pipet tip into the solution to be transferred. g. SLOWLY release the plunger to retrieve the liquid. h. Insert the pipet tip into the desired destination tube. i. Press the plunger past the first stop to the SECOND stop to transfer the liquid. Keep the plunger pressed when lifting the pipet tip out of the tube. j. Eject the pipet tip into the trash container.

_____ Secure one small foam “ice bucket” and fill it with ice.

_____ If your instructor has already aliquotted the loading dye and DNA samples into labeled micro test tubes for your team, you may skip the next 7 steps on this instruction check-list.

4 _____Secure 4 micro test tubes (one of each color) for your team. With a permanent marker, label the top of the tubes with one capital letter as follows: Yellow tube, L for uncut lambda DNA Purple tube, P for Pst1 lambda digest Green tube, E for EcoR1 lambda digest Orange tube, H for Hind lll lambda digest

_____ Secure one clear or light blue micro test tube for your team. Label the top of the test tube with LD. This will hold your loading dye which will make your DNA samples easier to see and will help the DNA sink to the bottom of the wells rather than float and spread away from the wells.

_____ While following every step of the correct micropipet procedures (see above), measure and transfer 12 microliters of loading dye from the stock supply into your clear micro test tube labeled “LD.”. Close the micro test tube and store it in a vertical position in your ice bucket.

_____ Take your micropipette, with a fresh tip attached, and the yellow “L” tube to the stock area. While following the correct micropipet procedures, transfer 10 microliters of uncut lambda DNA into your yellow micro test tube. Close the micro test tube and store it in a vertical position in your ice.

_____ Following correct procedure, transfer 10 microliters of Pstl restriction digest of lambda DNA into your “P” purple micro test tube. Close and store tube in your ice bucket.

_____ Transfer 10 microliters of EcoRl restriction digest of lambda DNA into your “E” green micro test tube. Close and store tube in your ice bucket.

_____ Transfer 10 microliters of Hindlll restriction digest of lambda DNA into your “H” orange micro test tube. Store tube in ice bucket.

_____ If any of the stock tubes of DNA appears to be empty, give the tubes to your instructor and he/she will centrifuge the tubes to force the liquid to the bottom of the stock tube where it can be retrieved.

_____ Adjust the micropipette volume to 2 microliters. It is very important to push the plunger of your micropipette only about 1 or 2 mm to the first stop to pick up the 2 microliters. If you go to the second stop of your micropipette, you will pick about 5 to 6 times more loading dye than needed.

_____ Transfer 2 microliters of your loading dye from your “LD” micro test tube into each of the 4 colored tubes containing your DNA. Use a fresh pipet tip for each transfer. (The loading dye will mix with the DNA and help it to sink into the wells as you load your gels.) As you finish with each micro test tube, close it, and store it in a vertical position in your ice.

_____ Take your micro test tubes (in their ice bucket) to the centrifuge area and then ASK YOUR INSTRUCTOR TO HELP YOU USE THE CENTRIFUGE MACHINE. If other teams are using the same centrifuge at the same time, mark your tubes so you can identify which tubes belong to your team. The tubes must be in a balanced symmetrical arrangement to prevent the destruction of the centrifuge machine. The spinning motion inside the centrifuge machine will force the loading dye and DNA samples to the bottom of the micro test tubes where the substances will mix.

5 _____ While following the correct micropipet procedures, measure and transfer 12 microliters of each sample into separate wells in the gel. To make sure that your tip is in the well, jiggle the tip back and forth slightly so that you can feel the sides of the well. Be careful not to pierce the bottom of the well because the DNA will leak out of the well. Load the gel in the following order: Lane Tube 1 L (yellow) 2 P (purple) 3 E (green) 4 H (orange)

_____ Other teams may need to share your gel. There are enough lanes for 3 teams to use one gel. Skip at least one lane between teams when sharing gels. Make a note as to which lanes belong to your team and which DNA is in each lane.

_____ CAUTION: STUDENTS, DO NOT TOUCH ANY PART OF THE Bio-Rad PowerPac Basic POWER SUPPLY. Your instructor will program the power supply and the instructor will connect it to the wall outlet later.

_____ CAUTION: STUDENTS, DO NOT CONNECT THE ELECTROPHORESIS CHAMBER TO YOUR POWER SUPPLY. Your instructor will connect all chambers to the power supply when all teams are ready.

_____WHEN ALL TEAMS HAVE FINISHED LOADING THE GELS, ASK YOUR INSTRUCTOR TO PLACE THE COVERS ON EACH CHAMBER AND TO START THE ELECTROPHORESIS PROCESS.

_____ CAUTION: STUDENTS, DO NOT TOUCH ANY PART OF THE POWER SUPPLY OR THE ELECTROPHORESIS CHAMBER WHILE THE POWER SUPPLY IS PLUGGED INTO THE WALL OUTLET. When necessary, your instructor will take that responsibility.

_____ We will be using 100 volts for 30 minutes for our electrophoresis activity. Record the starting time. If working properly, you should see tiny bubbles rising in both reservoirs of your electrophoresis chamber. Notify your instructor if there are no bubbles.

_____ While the DNA and loading dye are moving through the gel toward the positive side of the gel chamber, YOU CANNOT SEE THE DNA but you can see where the loading dye is migrating across the gel.

_____ After 30 minutes, your electrophoresis is complete. ASK YOUR INSTRUCTOR TO: (A)TURN OFF THE POWER SUPPLY SWITCH that is found on the side of the machine, (B) DISCONNECT THE POWER SUPPLY FROM THE WALL OUTLET, (C) UNPLUG THE RED AND BLACK CORDS (LEADS) FROM THE POWER SUPPLY, AND (D) REMOVE THE LID FROM THE ELECTROPHORESIS CHAMBERS.

_____ Hold the gel tray level as you remove it from the chamber. Because the gel is very slippery, you will need to support the sides of the tray to prevent the gel from sliding off of the tray prematurely.

6 _____ Gently slide the gel into the staining tray provided by your instructor.

_____While part of your team is following the staining directions, the rest of the team will follow the clean-up instructions which are the last 4 steps on this checklist.

_____ The “fast blast” stain molecules will bind to the DNA fragments and allow the DNA to become visible. IMPORTANT: USE CAUTION, WEAR GLOVES AND LAB COAT WHILE HANDLING STAIN TO AVOID STAINING SKIN AND CLOTHING.

_____ For quick staining your gel, pour just enough 100x Fast Blast stain into your staining tray to cover your gel. Immediately record your starting time. Gently agitate the gel for exactly 2 minutes and then QUICKLY pour off the excess stain. Return the excess stain to the Fast Blast container for reuse. The gel will remain in the staining tray for the destaining process.

_____ IMMEDIATELY begin the destaining process by rinsing the gel with cool or lukewarm water. Record the starting time. Continue rinsing for at least10 minutes while replacing the water frequently with fresh water. You may store your gel overnight in a plastic bag with about 100 ml of water to further destain the gel.

_____ After rinsing and destaining the gel, allow all team members to observe the gel.

_____ If your instructor has gel support film for you to use, secure one piece of film and ask your instructor how to trim away any unused areas of your gel. Remember to handle the gel carefully because it is very fragile. After trimming, place the gel directly upon the hydrophilic side of your gel support film. (Water will form beads on the hydrophobic side but will spread flat on the hydrophilic side of the film.) Center the gel on the film and place the film on a paper towel in the dark to dry. Label the paper towel with name and date. The gel will dry completely in 2-3 days and shrink in size.

_____ If your instructor does not want to use gel support film, you will want to store your gel in a resealable quart sized plastic bag. With a permanent marker, write your names on the bag. Carefully transfer the fragile gel into the bag. Add a small amount of water. You may store the gel overnight at room temperature or for several weeks in the refrigerator.

_____ Return your used TAE buffer solution from your electrophoresis chamber into the TAE container for reuse.

_____ Rinse the gel tray and electrophoresis chamber with water and then clean them with a paper towel dampened with 70% alcohol solution.

_____ Clean the surface of your work area with 70% alcohol solution and paper towels.

_____ Use your “Workstation Inventory” to make sure that all supplies are in your work area now. Notify instructor if something is missing. Give your “Workstation Inventory” to your instructor at this time.

7 Precut Lambda Lab STUDENT WORKSTATION INVENTORY

Beginning End Items per team: 1 workstation inventory (check this at 1st & end of lab) 1 instruction manual and or checklist per student Paper towels 1 micropipet 1 container of micropipet tips 1 small container for used pipet tips & trash 1 small foam ice “bucket” with ice (DO NOT EAT this unsafe ice) 1 set of 5 snap cap microcentrifuge tubes (preloaded or ready to load) 1 foam micro test tube holder 1 pair goggles per student (clean with alcohol prior to use) 1 pair gloves per student

Beginning End Items to share with another team: 1 staining tray 1 permanent marker (to label plastic bag with names/date) 1 quart size resealable plastic bag (to store gel after staining) 1 micropipet stand 1 electrophoresis chamber with lid 1 gel tray 1 prepared gel (unless you are making your own) If you are making your own gel, you will need the following additional items: 2 dams or “casting gates” per chamber 1 gel comb with 15 “teeth” 1 leveling bubble 1 flask 125mL

Items to share with entire class: 1 microcentrifuge machine Agarose powder (if students are making gels) 1 power supply 100 mL graduated cylinder (If students are making gels.) TAE buffer for electrophoresis chambers Scales or balance to measure agarose powder (if students are making gels) Fast Blast stain Chemical scoop (if students are making gels) Weighing paper (If students are making gels.) Stock tubes of DNA (if not already aliquotted) Microwave oven (if students are making gels) Stock tube of loading dye (if not already Hot pads (if students are making gels) aliquotted)

Captain and team members:

Team Comments:

8 At the beginning and end of the lab period, please check the workstation inventory and report any missing items to your instructor immediately. Turn in this form at the end of the period today.