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BLOCK 5-2 week-6 GENERAL TOPIC: genetics testing OBJ: 11-12 DO NOW: NONE EXT: DUE DATE: DW: 13.2 LAB -DW13.2 VIRTUAL LAB -ELECTROPHORESIS LAB ABSENT AGENDA ------------------------------------------------------------------------------HANDOUTS to PICK-UP: -13.2 LAB TURN IN to ABS box: SEMINAR 2: BEFORE/AFTER SCHOOL: DW: 13.2 VIRTUAL LAB EXIT:CIRCLE-level of understanding of today’s objective ON BACK NONE BLOCK Write this in your Weekly Organizer in RED PEN LEVEL OF UNDERSTANDING 13.2 VIRTUAL LAB 5 DW GRADE __________ / __________ 13.2 VIRTUAL LAB-GRADE RUBRIC 5-WHAT IS EXPECTED -All areas COMPLETED 3-ALMOST WHAT IS EXPECTED -50% COMPLETED 0-NOT COMPLETED GEL ELECTROPHORESIS 1-What is your job? -determine lengths of DNA 2-IDENTIFY the FUNCTION of GEL ELECTROPHORESIS? -SORT & MEASURE DNA strands by length 3-How does GE work? -gel acts as a filter -separates molecules (electrical charge) -sorts DNA strands (banded pattern) SEQUENCE steps of DNA moving thru gel GEL ELECTROPHORESIS 4-Do LONG or SHORT strands move more quickly thru the gel? -SHORT 5-Can we see single strands of DNA in the gel? -NO—they are too small even under a microscope GEL ELECTROPHORESIS 6-LIST 5 basic steps of GE -______________________________________ Make gel -______________________________________ Set up apparatus -______________________________________ Place DNA samples in holes at end of gel ADD electric current -______________________________________ -______________________________________ Stain-ANALYZE MODEL 5 steps of GE with picture procedures GEL ELECTROPHORESIS WALK THRU LAB 7-DETERMINE what each is made of AGAROSE: -seaweed BUFFER: -salt water solution 8-why do buffer & agarose need to be heated? -melt agarose into buffer 9-why does the mold have tape on each end? -holds melted agarose 10-IDENTIFY why a COMB placed in the gel at one end -makes “wells” (small holes) to hold DNA samples 11-ANALYZE 2 reasons buffer solution is added to GE box -conducts electric current -keeps gel from drying out GEL ELECTROPHORESIS 12-DETERMINE 2 functions of loading buffer -contains a dye making sample easy to see -makes DNA sample thicker so it doesn’t float away 13-PREDICT in a real lab difficulties loading samples -NOT easy—might take some practice 14-why would we use a standard DNA size sample? -contains DNA strands of known lengths -provides reference to estimate lengths of DNA strands 15-CONCLUDE why electricity is used to run the wells -DNA negative charge attracted to + charge GEL ELECTROPHORESIS 16-how can you check to make sure your current is running? -should see tiny bubbles 17-does DNA move to + or – end? -+ 18-why is the gel stained and how does it work? -cannot see DNA but can see dyes -can see large groups of stained DNA strands -dye binds to DNA-shows up under flouresent lights GEL ELECTROPHORESIS 19-ENTER bp— base pairs —size estimates -6000 -3500 -1500 13.2 LAB OBJECTIVE: -student will DEMONSTRATE the separation technique known as gel electrophoresis. -student will use this process to IDENTIFY dye samples by molecular mass. -student will use this information to IDENTIFY the composition of an UNKNOWN mixture of dyes found at crime scene 13.2 LAB INTRO/ BACKGROUND: -How can a mixture of molecules, too small to be seen with even a high-powered microscope, be separated from one another? Such was the dilemma facing scientists until the development of a process that has now become standard in many laboratories worldwide- gel electrophoresis. -Laboratories rely heavily on this proven and reliable technique for separating a wide variety of samples, from DNA used in forensics and for mapping genes, to proteins useful in determining evolutionary relationships. -In 1950 gel electrophoresis was invented. The process involves applying an electrical current to a gelatin-like substance containing biological samples. When mixtures of materials are placed within the wells of the gel and an electric current is applied, the molecules travel through the gel and separate from one another according to each molecule’s charge, size, shape. The gel acts as a type of molecule strainer and prevents all the molecules from moving too quickly. Smaller molecules typically mover through the gel at a faster rate than the larger ones. When the current is turned off, all the molecules are stopped within the gel. If a dye is added to the samples placed in the wells, individual groups of molecules can be identified by a distinct, colored band within the gel. When the distance each band (group of molecules) traveled is measured and compared to the other colored bands within the gel, the measurement can readily distinguish smaller molecules from larger molecules 13.2 LAB PROBLEM: Yesterday, there was a home break-in. No fingerprints were found at the scene but forensic investigators found multiple threads of what appears to be clothing. The thread’s colored was unable to be determined as it appears the clothing was also slightly burned, thereby affecting the ability to see the color. Since many clothing dyes are made from plants, the DNA can be extracted from the dye of the cloth and matched with the crime scene thread. Here is the list of suspects: 1-Dereck Simien – 49, Male, wore a green t-shirt at the time of the robbery 2-Jamie McDonald – 33, Female, wore an burnt orange hoodie at the time of the robbery 3-Martha Dougal – 34, Female, wore a blue denim coat at the time of the robbery 4-Louis Castillon – 20, Male, wore a red jacket at them time of the robbery 5-Scott Hackman – 25, Male, wore a K-State polo at the time of the robbery GEL ELECTROPHORESIS CLEAN 1- Gather needed materials and make sure working area is ______________ and ____________. DRY HORIZONTAL 2- Place the electrophoresis unit in a ____________________ position on the countertop. 3- Place the “gel drawing worksheet” on the counter next to or below the electrophoresis unit. LEFT to #6 at the __________. RIGHT 4- Number the wells on the paper from #1 at the _______ 6 5- Withdraw ________microL of dye from each tube by filling only the needle tip of the pipet. 6- USE A CLEAN PIPET FOR EACH DYE SAMPLE TO AVOID CONTAMINATION. 7- Make sure you follow the data table for the correct dye in each numbered well. 8- Once all dyes have been transferred to the wells, NOTIFY YOUR TEACHER-The TEACHER will load your gel into the electrophoresis unit. PART 4: Testing for the HD gene LOADING INSTRUCTIONS: GETTING THE SAMPLE PART 4: Testing for the HD gene LOADING INSTRUCTIONS: LOADING THE GEL 1 2 3 4 5 6 13.2 LAB PRE-LAB Q’s: -Using the information in the chart / background knowledge answer the following questions. 1-PREDICT the DIRECTION of migration for each unknown dye sample. (+ or -) GREEN + ORANGE + BLUE + RED + PURPLE + 2-PREDICT which dye will move through the gel the: FASTEST ______________ SLOWEST ______________ 13.2 LAB PRE-LAB Q’s: 3-LIST a BRIEF FUNCTION for each part used in gel electrophoresis AGAROSE GEL-”microscopic strainer” / separates BIG molecules from SMALL BUFFER-conductor / carries electric current WELLS IN GEL-hold the DNA sample ELECTRIC CURRENT-provides force that MOVES the sample 13.2 LAB 15- Use the [GEL DRAWING] template to make an accurate drawing of the bands in the gel. -USE COLORED PENCILS 16- Measure the distance each gel migrated in mm. Record in data table.[DATA TABLE] 17- Record the charge for each dye molecule by noting which pole (+/-) the dye sample was traveling when the power was shut off. [DATA TABLE] 18- Use the recorded migration distances to complete the last column of the data table by ranking the five known dye samples from fastest (#1) to slowest (#5) [DATA TABLE] 19- CLEAN-UP lab station. _____- dispose of gel in trash can _____- rinse AND dry petri dish (return to cart in front of room) _____- wipe down lab station _____- return all equipment to original location. [see board for reminder of location]