S-STEM Scholar Zaira Grijalva's Blog: Week 2. Semester 2. First DNA extraction a Sucess!!

This week I had the opportunity to extract DNA from e. coli under Paul's supervision and guidance. Although his method is pretty much fool proof I was super excited to see the banding on our gels! I honestly though my first time would be a disaster hehe. The protocol followed is summarized below.

1. Centrifuge 1mL of cultured TSB @ 12000 rpm for 3 minutes.

2. Discard supernatant (broth) & add 0.3 mL TBS & 15 µL 10% SDS to each tube.

3. Incubate @ 55ºC for 10 min.

4. Cool to room temp

5. Add 5µL proteinase K - Vortex 20 sec.

6. Incubate in ice for 5 min.

7. Centrifuge @12000 rmp for 3 min. (to precipitate proteins)

8. Transfer supernatant to clean 1.5 mL tubes.

9. Add 300µL room temp isopropanol.

10. Invert until DNA forms threads.

11. Centrifuge @ 12000 rpm – 2 min.

12. Discard supernatant. Add 300µL room temp 70% ethanol. Invert to wash pellet.

13. Centrifuge @ 12000 rpm – 2 min.

14. Discard supernatant & air dry DNA (we used the hood for 20min.)

15. Resuspend DNA in sterile water (50µL).

While the samples were incubating for step 3 above, we prepared 1xTAE buffer by adding 980 mL of di water to 20 mL of TAE 0.5%.

While DNA was drying under the hood, we prepared the gels for electrophoresis by adding 0.9 g of agarose to 100mL of 1x TAE buffer. We proceeded to microwave this beaker for 30 sec intervals until diluted. Once it cooled down to 59ºC we added 4µL of cyber green.

We then poured 25mL of agarose/TAE buffer solution into the gel molds we had set up previously and covered with box to keep in the dark since cyber green is very light sensitive.

Once the gels were set and the DNA had dried, we completed step 15 of the above protocol and proceeded to load 4µL of DNA sample and 2µL of loading dye in the wells in the gel.

The last step was to run electricity (120V) through the gels for approximately 15 min. And view them under UV light. It was a success!! Please see pictures below.

Thank you very much to Paul Cattelino for your help and all the little tips you shared with me! I love being part of this program and feel honored to being able to work alongside and collaborate with you!!