WET LAB DNA Barcoding: From Samples to Sequences - Northwest ...

More documents

Recommendations

Info

WET LAB d. Ask students to estimate the size of the sample bands in the sample lanes (Lanes 2–5). These bands are just below the 750 bp band in the molecular weight standard lane, and are estimated to be between 650–700 bp. [Note: These are PCR samples from a DNA barcoding experiment with salmon.] e. Ask students to estimate the amount of DNA in the sample lanes, using the known quantity of DNA in the molecular weight standard lane that is closest in size to the DNA sample bands (i.e., 750 bp, 25 nanograms (ng) of DNA). • Lane 1: Approximately 25 ng. The band is about the same intensity as the 750 bp band in the molecular weight standard lane. • Lane 2: Approximately 50 ng. The band is about twice the intensity of the 750 bp band in the molecular weight standard lane. • Lane 3: Approximately 75 ng. The band is about three times the intensity of the 750 bp band in the molecular weight standard lane. • Lane 4: Approximately 100 ng. The band is about four times the intensity of the 750 bp band in the molecular weight standard lane. • Lane 5: Approximately 125 ng. The band is about five times the intensity of the 750 bp band in the molecular weight standard lane. 32. Show Slide #20, which illustrates some of the different methods used to visualize DNA gels: ethidium bromide with UV (ultraviolet) light or Fast Blast blue stain (from Bio-Rad Laboratories) with visible (white) light. While the methods may differ, the results are the same: the stain binds to the DNA in the molecular weight standard and sample lanes on the gel, and then the stain is visualized by shining light through the gel. Wet Lab: Slide #20 Using Bioinformatics: Genetic Research 33. Pass out Student Handout–Analyzing PCR Results with Agarose Gel Electrophoresis. Have students work through the activity in the same small groups from Lab 2. 340 ©Northwest Association for Biomedical Research—Updated October 2012
WET LAB 34. Optional Graphing Extension Activity: Students can graph the log of the molecular weight of each band in their molecular weight standard (y-axis) against the distance each band traveled, in centimeters or millimeters (x-axis), either by hand or in a graphing program like Microsoft ® Excel. Students can then determine the exact molecular weight of their PCR product band(s) by measuring the distance traveled by their DNA bands, and either plotting it on their graph or using their regression equation to solve for Y (molecular weight). Lab 4: Preparation of PCR Samples for DNA Sequencing Teacher Preparation • Load the classroom computer with the Wet Lab PowerPoint slides. • Make copies of the Student Handout—Preparation of PCR Samples for DNA Sequencing, one per student. These handouts are designed to be reused as a class set; students should write answers to questions and take notes on a separate sheet of paper or in their lab notebook. • Teachers may wish to have their students write out the lab procedures before the activity in their lab notebooks or on a separate piece of paper as a “prelab” exercise, which can be used as an “entry ticket” to class. During the lab, students may check off steps as they complete them and/or describe and draw pictures of their observations. • Queue your computer to one of the following tutorials on DNA sequencing: ‣ “The Sanger Method of DNA Sequencing” by the Wellcome Trust. Freely available at: http://www.wellcome.ac.uk/Education-resources/Teaching-and-education/ Animations/DNA/WTDV026689.htm. ‣ “Sanger Method of DNA Sequencing” video freely available from the Howard Hughes Medical Institute (HHMI). Video is 51 seconds long and requires an internet connection and speakers. Available at: http://www.hhmi.org/biointeractive/dna/DNAi_sanger_sequencing.html. • Set up student work stations by distributing supplies and reagents needed for each group, as described above under Materials. It is suggested that reagents from the stock bottles contained in the DNA Clean & Concentrator-5 (capped columns) kit be aliquoted for student groups and labeled as described in Student Handout—Preparation of PCR Samples for DNA Sequencing. This information is found in Teacher Resource—Aliquoting DNA Barcoding Reagents for Labs 1–4. 35. Show Slide #21, “Preparation of PCR Samples for DNA Sequencing,” as students enter the room. Wet Lab – DNA Barcoding: From Samples to Sequences 341 ©Northwest Association for Biomedical Research—Updated October 2012
Page 1 and 2: WET LAB DNA Barcoding: From Samples
Page 3 and 4: WET LAB Lab 1: DNA Purification for
Page 5 and 6: WET LAB Lab 4: Preparation of PCR S
Page 7 and 8: WET LAB • Teachers may wish to ha
Page 9 and 10: WET LAB Wet Lab: Slide #3 Lysis or
Page 11 and 12: WET LAB • If students are not fam
Page 13 and 14: WET LAB Wet Lab: Slide #9 14. Show
Page 15 and 16: WET LAB Lab 3: Analyzing PCR Result
Page 17 and 18: WET LAB 20. Show Slide #12, and rem
Page 19 and 20: WET LAB Wet Lab: Slide #15 26. Tell
Page 21: WET LAB Wet Lab: Slide #18 Wet Lab:
Page 25 and 26: WET LAB e. Deoxynucleotides (dNTPs)
Page 27 and 28: WET LAB Glossary Agarose gel electr
Page 29 and 30: WET LAB PCR beads: Lyophilized or
Page 31 and 32: WET LAB CLASS SET Lab 1: DNA Purifi
Page 33 and 34: WET LAB CLASS SET Day 2 Procedure:
Page 35 and 36: WET LAB CLASS SET 14. Place the spi
Page 37 and 38: WET LAB CLASS SET Lab 2: Copying th
Page 39 and 40: WET LAB CLASS SET 9. Add each of th
Page 41 and 42: WET LAB CLASS SET Lab 3: Analyzing
Page 43 and 44: WET LAB CLASS SET 11. To prepare yo
Page 45 and 46: WET LAB CLASS SET Lab 4: Preparatio
Page 47 and 48: WET LAB CLASS SET On your separate
Page 49 and 50: WET LAB KEY Lab 1: DNA Purification
Page 51 and 52: WET LAB KEY Lab 2: Copying the DNA
Page 53 and 54: WET LAB KEY Lab 4: Preparation of P
Page 55 and 56: WET LAB RESOURCE Aliquoting DNA Bar

WET LAB DNA Barcoding: From Samples to Sequences - Northwest ...

Create successful ePaper yourself

Delete template?

Save as template?