Exp 7 wk2 - Procedure & Report Info for Exp 7

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Mohawk College *

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10003

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Biology

Date

Feb 20, 2024

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pdf

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Uploaded by CoachGrousePerson817

Page 1 of 3 Week 2 of Exp #7 (Performed with Exp #8 wk 2) Week 2 of Exp 7 – Running Out Diagnostic Digest Week 2 of Exp 7 will be carried out in the same lab period as week 2 of Exp 8. Refer to ‘Exp 7 & 8 Wk 2’. PROCEDURE 1. Remove your PstI diagnostic digests, PCR samples, and DNA size marker from freezer. 2. Weigh 0.8 g of agarose powder, and place in a beaker. Add 100 ml of 1X TBE buffer. Microwave on medium for ~2 min until clear. Swirl the hot solution using hot gloves. Make sure the agarose is completely dissolved. 3. Immediately add DyeNA-View gel stain (amount as directed on side of tube). Swirl beaker to mix. 4. Assemble the larger sized gel electrophoresis equipment. Set up a try that is inserted into the gel box such that all sides are sealed. Place a 14-well come in the tray. Adjust the comb holder to the correct height. 5. When the agarose solution with DyeNA-View gel stain has cooled sufficiently, pour the liquid gel into the tray with the14-well comb. 6. Wait ~20 minutes until gel is solid. 7. Take the tray with the solid gel out of the gel box and rotate it 90º so that now the ends of the gel are exposed to the TBE buffer that will be poured into the gel box. 8. Pour 1X TBE buffer into the gel box. Make sure the wells of the gel are entirely submerged in buffer. 9. Hold the tray down with your hands, and gently remove the comb while the gel is submerged in buffer. (You can have your partner remove the gel comb if this is easier for you.) 10. In your lab notebook write Lane 1 2 3 4 5 6 7 on one row. Skip a few lines . Beneath write Lane 8 9 10 11 12 13 14 Write down which samples you will load in which lanes. Lane 1 should have DNA ladder. (The volume of DNA ladder that you load depends on the specific DNA ladder used. Check with your instructor for detailed instructions.) You need to know the specific name of the DNA ladder (marker) you are using, and the company from which it originates so copy this information from the DNA marker tube used. Lane 2 should be blank. Lanes 3 and 4 should have the PCR reactions. Lanes 5 should be blank. Lanes 6 – 11 should have your six PstI diagnostic digests. Lane 12 should be blank.

Page 2 of 3 Lane 13 should have DNA ladder. Lane 14 should be blank. (Modify loading and lane designations as necessary.) 11. Load the DNA ladder and samples (entire volume) as outlined above. Use fresh tips between loading each sample. 12. Connect the lid and the power leads to the power supply. 13. Set the power supply to 140 volts. Run the gel for as long as is required to achieve good separation of bands (at least 30 min; ideally longer). 14. Turn off the power supply and remove the lid. Pour off the buffer. 15. Transfer the gel carefully onto the U.V. light box. 16. Use the gel doc system to visualize and take 2 pictures/person of the gel (one for Exp 7 report and one for Expt 8 report). 17. Once you have your photographs, make a chart of: i) Size of each DNA ladder band in base pairs ii) Distance migrated by each DNA ladder band from well in cm iii) Distance migrated by each band in your PstI diagnostic digests iv) Distance migrated by each band in your PCR bands. This chart must be included in the ‘Results’ section of BOTH your Exp 7 and Exp 8 reports. POST LAB – INDIVIDUAL FORMAL REPORT A formal lab report is required for this lab and must be submitted individually to the “Exp #7” folder on Canvas one week after the completion of this experiment. Must include: Title page, Introduction, Materials/Methods, Observations, Results, Discussion/Conclusion, Post-Lab Questions, and a copy of your lab notebook initialed by your instructor. You must submit the report individually. Graphing a Standard Curve Refer to the data from step 17 i) and ii) in your chart. Using a semi-logarithmic graph template, plot the size of each band in base pairs on the y-axis (i.e. the log scale) vs. the distance migrated by each band from the gel well in cm on the x-axis (i.e. the non-logarithmic axis). Connect the points with a straight line. This will create a standard curve . (You can simply photocopy, or print two copies of the standard curve to use for Exp 7 and Exp 8.) Estimating Size of Bands in Diagnostic Digests Refer to the data from step 17 iii) in your chart. Mark the distance migrated by each band (in cm) in each of your diagnostic digest lands on the x-axis of the standard curve, and then read across to the y-axis to determine the size in base pairs of those bands.

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