User:Daniel-Mario Larco/Notebook/AU Photosynthesis Lab/2014/03/26

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Objective

During this lab session, solutions of LB/Agar and LB Broth were prepared. A solution of Ampicilin was prepared to be added to the broth and agar. Sequence preparation was also done to order plasmid DNA containing Hemoglobin

Sequencing

  1. 4 sequences of hemoglobin were provided by Dr. Hartings
    1. WT Ascaris Hemoglobin
    2. T109C NoCys Hemoglobin
    3. K31C NoCys Hemoglobin
    4. K31C T109C NoCys Hemoglobin
  1. To each of the sequences, a start ATG and stop TAG codon were added
  2. Between the start codon and the start of the sequence, 6 Histidine codons CAT were added
  3. The PacI restriction enzyme targeted sequence was added before the start ATG and after the stop TAG codons for each sequence.
    1. For the first set of sequences the entire restriction sequence was added before and after
    2. For the second set of sequences only the over hangs of the restriction sequence was added
      1. This required finding the reverse complimentary sequence for each type of Hb in order to add the over hangs for the 3'->5' direction

Procedure

1. The correct concentration for dissolving LB Broth Media into water is 25g per liter of water. The correct concentration for dissolving granulated Agar into water is 20g per liter of water.

- For the LB/Agar solution. 2.51g of LB Broth Media and 2.00g Difco (TM) granulated Agar were dissolved in 100mL of water

The solution is placed in the autoclave at 121 degrees C for an hour.

2. In order to prepare the ampicilin solution, 100mg of Ampicilin Sodium Salt were dissolved into 1mL of sterilized water. This solution was prepared in a sterilized eppendorf tube using sterile equipment.

3. Once the solutions cool, 100uL of the ampicilin solution is added to the LB/Agar solution

4. The LB/Agar solution is now plated. approximately 25mL of the solution is poured into the sterilized plates.