User:Elizabeth Ghias/Notebook/Experimental Biological Chemistry/2011/09/20

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Objective

To mutate GFP through PCR so that it contains a cysteine residue after the enterokinase cleavage site.

To prepare starter culture media and protein expression culture media for protein expression.

Description

PCR

  1. The PCR quick change manual was used to determine which forward and reverse primers should be used for the PCR. Prepared primers were used for the PCR reaction
  2. A reaction mixture was prepared with the following contents: 1 μL of template DNA, 1.25 μL of primer 1 (D1C F), 1.25 μL of primer 2 (D1C R), 5 μL of 10X Pfu reaction buffer, 1 μL of dNTP mix, and 1 μL of PfuTurbo DNA polymerase.
  3. 25 μL of Bio Rad Chill Out Wax was placed on top of the reaction mixture
  4. The reaction mixture was placed in the thermocycler for one cycle at 95°C for 30 seconds
  5. The reaction mixture was kept in the thermocylcer for 16 cycles with the profile shown below.
  6. The reaction mixture was left in the thermocycler for 24 hours at 4°C
       PCR Profile
   1) 95 °C for 30 seconds
   2) 55°C for 1 minute
   3) 68°C fr 3.6 minutes


Protein Expression Preparation

Starter Culture Media:

  1. In a 250 mL flask, 0.875 g of Lysogeny (Luria) Broth and 35 mL of water were mixed. Four flasks were prepared with this mixture.
  2. The flasks were covered in foil and autoclaved for 50 minutes at 121 F.
  3. Once the flasks cooled, 35 μL of 1000X ampicillin antibiotic was added to each flask. The ampicillin was prepared by mixing 0.450 g ampicillin with 4.5 mL H2O.
  4. The flasks were then inoculated with a single colony of bacteria using sterile methods.
  5. The flasks were placed in a incubator/shaker set to 37°C and 200 rpm. The flasks were left in the shaker/incubator overnight.

Expression Culture Media:

  1. In a 2.8 L Fernback flask, 25G or Luria Broth and 1 L of water were mixed. Four flasks were prepared in this manner.
  2. The flasks were covered in foil and autoclaved for 50 minutes at 121 F.
  3. 1000X antibiotic was prepared by mixing 0.450 g ampicilin 4.50 mL of water.
  4. Once the flasks had cooled, 1 mL of 1000X antibiotic was added to each of the four flasks.
  5. 4 mL of 0.1 M isopropyl β-D-1 thiogalactopyranoside (IPGT)was also prepared for use the following day.

Data

  • Add data and results here...

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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