User:Elizabeth Ghias/Notebook/Experimental Biological Chemistry/2011/09/21

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Objective

To preform electrophoresis using an agarose gel to determine if the PCR performed the previous day successfully mutated the GFP protein.

To carry out protein expression using the starter culture media and the expression culture media prepared the day before.

Description

Electrophoresis Procedure

  1. A 1% agarose gel was prepared using 0.25 g agarose and 25 mL of 1X TAE buffer.
  2. The flask containing the agarose and 1X TAE buffer was microwaved for 40 seconds.
  3. The gel casting tray was assembled and the comb was placed in the tray.
  4. The agarose solution was poured into the mold and allowed to sit until it had cooled and solidified.
  5. Once the gel had solifified, the comb was gently removed.
  6. The gel was placed on the center of the electrophoresis chamber.
  7. The reservoirs surrounding the gel were filled with additional TAE buffer. The gel was submerged in buffer about one centimeter.
  8. The DNA samples were prepared for the gel by mixing 1 μL of loading dye with 5μL of PCR product.
  9. 6 μL of each sample was added to each gel wall, beginning with a DNA ladder in the first well on the left.


Protein Expression

  1. The starter culture was centrifuged at 4500 rpm for 15 minutes.
  2. The cell pellets were resuspended in 4 mL of Luria Broth.
  3. The resuspended cells were divided between the four Fernbach flasks to inoculate the expression culture media.
  4. The expression cultures were incubated at 37°C until their absorbance at 600 nm equaled 0.6.
  5. 1 mL of the IPTG prepared the previous day was added to each flask.
  6. The flasks were shaken for 2 hours.
  7. Two of the flasks were spit into 2 750 mL plastic containers with caps, so that each plastic container had the same mass of expression culture.
  8. The four of the plastic containers were centrifuged for 15 minutes each at 4500 rpm.
  9. The fluid was emptied out of the centrifuge containers, leaving the cells in the bottom of the four containers.
  10. The contents of the remaining two Fernbach flasks were spit between the four plastic containers containing the cells.
  11. The containers were centrifuged again for an additional 15 minutes at 4500 rpm.
  12. The fluid from the flasks were removed, leaving cells at the bottom of the containers.
  13. 35 mL of 25 mM Tris and 50 mM NaCl (filtered to pH 8) was added to two of the containers. Once the cells were dissolved, the volume added was transferred to the next two containers.
  14. The dissolved cells were then transferred to two plastic tubes and stored at -80°C for one week.


Data

Image:DNA_gel_092111_003.jpg

From the left, the lanes are the ladder, group 1, 2, 3, 4, and 5

Notes

This area is for any observations or conclusions that you would like to note.


Use categories like tags. Change the "Course" category to the one corresponding to your course. The "Miscellaneous" tag can be used for particular experiments, as instructed by your professor. Please be sure to change or delete this tag as required so that the categories remain well organized.



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