Holli Brown

CILI-CURE 21

2/14/19

Objectives:

The objectives for this lab were for students to successfully purify DNA samples which had been incubating in the freezer since last lab. Students will also practice various new and important techniques in DNA purification, such as using the vacuum chamber and carefully handling harmful substances such as Ethidium Bromide. Students will also work cohesively as a group, and delegate different duties throughout lab, in order to efficiently finish the entire lab. Lastly, students will also use critical thinking skills and equations to solve problems of dilution and concentration, in order to make the agarose gel correctly. Overall, students will prepare for the gel electrophoresis phase of next lab, and correctly create purified samples and agarose gel, all whilst practicing safe and aseptic techniques.

Methods and Lab Summary:

1. Remember to have gloves when handling supplies in lab today. We want to avoid the transfer of human DNA to the sample, as well as avoid corrosive and mutagenic chemicals getting on the skin.
2. We will start with the crude DNA purification. Obtain 1 mL of the crude supernatant from the refrigerated Eppendorf tube from last lab. Micropipette this solution into a 15 mL tube.
3. To the tube, add 2mL. of warm DNA resin, and mix by swirling and inversion.
4. Obtain a syringe and a column and assemble it to where the column fits on the end of the syringe. Move your work station to the vacuum chamber underneath the fume hood.
5. Transfer around 1.5 mL of DNA resin mixture at a time into the syringe and column apparatus. Make sure the vacuum is on, and the column fits snugly into the vacuum holes. Add the solution in increments, so that the system doesn’t get clogged.
6. After all 3 mL of mixture are properly ejected into the vacuum system, attain the isopropanol alcohol. The alcohol should be stored in a 15 mL tube, in which we will use 6mL altogether. Again, be sure to use incremental measurements when putting the liquid into the syringe, such as 0.5 mL per ejection.
7. Eject all 6 mL into the syringe. Note that with time, the rate at which the liquid will be sucked up will slow down.
8. After the vacuum step has been completed, transfer the column into an Eppendorf tube, removing the syringe part and discarding of it.
9. Centrifuge the Eppendorf tube on 8,000 RCF for 5 minutes, making sure the centrifuge is balanced. We do not want to break this expensive piece of lab equipment!
10. After 5 minutes of centrifugation, transfer the column out of the tube and into a heat block for 30 seconds, at around 80 degrees C. Afterwards, put the column into a new tube, and discard the old one, as there could be left over alcohol in it.
11. Add 50 uL of warm, sterile DI water into the tube and column, and let this incubate for one minute.
12. Centrifuge this mixture for 1 minute at 8,000 RCF again. Throw away the column, and keep the Eppendorf tube. Be sure that your tube is labelled, and store it within lab. The stuff inside the tube is our purified DNa, now ready for electrophoresis.
13. For the agarose gel, set down measuring paper on an analytical scale. After it has been zeroed, add 0.4 g of agarose powder.
14. Obtain a 50 mL seafood green capped tube, and funnel the powder in, keep the measuring paper.
15. Next, add 2 mL of Tris-EDTA-Acetic acid, or TAE into the 50 mL tube along with the powder. Be sure to do the calculations right, because we want a 1x solution here.
16. Add DI water up to the 40 mL line, and mix by inversion, as the powder can get stuck on the bottom of the tube.
17. Transfer to solution into an Erlenmeyer flask, and cover it with the measuring paper. Transfer to the LA and have them microwave it for 1 minute.
18. Afterwards, let the gel solution cool for 5-6 minutes, an indicator of appropriate temperature is to wait until the bottom doesn’t burn the palm of your hand.
19. Bring the solution over to the LA. They will add a hazardous staining chemical called Ethidium Bromide. This solution is a carcinogen, and can cause mutations within cells. They will add 2 uL of this solution into the flask, and mix it by swirling it.
20. Assemble a mold for the gel to be placed in, and make sure it is water tight. Also be sure to put the apparatus that will create the wells facing the right way, the white part toward the larger part of the gel interface.
21. Pour the agarose gel into the mold, and allow it to set. Label it with your group members name and class number.
22. Clean out the flask, as we do not want agarose gel forming within the flask as well. Clean up your lab bench, and finish the QTM if time allows.

Data and Observations:

Below is an image of the supernatant in the 15 mL tube without resin. We wondered why the substance was a dark orange brown color, and we though maybe it was due to the DNA Extraction media added last lab.

Below, is an image of the vacuum apparatus like the one in seen in lab. The column fits into one of the small holes, and as the person is in the image below, liquid would be pipetted into the syringe.

Below is a picture of the mold and the agarose gel in the Erlenmeyer flask. The gel when solidified turned a clear milky color.

Storage:

The agarose gel was labelled and kept in the container, and was taken up by the LA. The eppendorf tube with the purified DNA sample was placed in a rack, and taken care of by the TA. The Erlenmeyer flask was thoroughly rinsed, and placed upside down to dry. The vacuum station was turned off, and materials were stored properly.

Conclusions:

In conclusion, we successfully purified our crude DNA through many new techniques learned in lab today.We were very good at delegating tasks, as Haleigh and Leslie mainly handled the DNA purification step, and while they were busy centrifuging, I started on the agarose gel. We were very efficient as we were the first group to complete the DNA purification, as well as our QTM’s. We pondered why the DNA would form lines within the agarose gel when electrophoresis is done, and we concluded that its because the DNA is attracted to the positively charged current end of the agarose gel, and it will change color due to the Ethidium Bromide. Overall, we communicated very well, and our samples hopefully tuned out successful. The agarose gel looks to be a good consistency, as its results could be seen at the end of lab because it has a fast solidifying time. Although the calculations at the start of lab were a bit challenging, overall we completed the experimental setup with little problems.

Future Steps:

In the future, I would have had a larger working space where the vacuum was, as it became crowded very quickly. I would also adjust the calculations to be more clear, because I felt as though I couldn’t have done it without a push from the LA and TA. Overall though, this experiment was relatively smooth.