Procedure for Isolation and Purification of Product (page 16 to 20)

Isolation ( Stage 1 )

10 mL of culture broth was collected in a tube.
The cells were centrifuged at 10,000 rpm for 5 minutes.
Supernatant obtained was transferred into another tube
UV light was used to confirm the product present in the pellet.

The product inside the pellet would go through three different methods of cell disruption.

Method 1: Using Enzymes

Using a micropipettor, the pellent was resuspended in 500 µL of TE buffer (pH 7.5) till no visible clumps were seen.
Two drops of lysoyme was added into the resuspended cell pellet using 1mL plastic pipette and left untouched for 15 minutes.

Method 2: Freezing and Thawing

The tube was placed into liquid nitrogen until the contents are frozen.
The tube was then thawed using warm water.
Repeat the cycle of freezing and thawing for another two times.

Method 3: Sonication

4 cycles of 25 seconds with 10 seconds rest in between sonication cycles were carried out on ice.
The content was then centrifuged for 20 minutes at 10,000 rpm.
The pellet and the supernatant were then separated.
The pellet was then resuspended using 400 µL of TE buffer.
The product (GFP) now would be inside supernatant and to be purified.

Steve doing the sonification(he looks like a DJ :D )<br>

Purification (Stage 2)

Gel permeation or size exclusion chromatography was used to purify the extract obtained from stage

Procedure
1. Nine test tubes were prepared on a rack.
2. The tubes were then labeled 1-8 and another a blank.
3. 2.0ml of ammonium bicarbonate was added to the blank tube, and a marking is drawn onto the 2ml level as well as for the other 8 tubes, for easy reference when collecting fractions.
4. The buffer in the column was allowed to drain off into the waste beaker until the buffer touches the top of the gel bed.
5. The bottom of the column is then capped.
6. Cell-free extract was then added onto the gel bed gently and evenly by using a dropper.
7. The column was uncapped and the media allowed to flow through, the first fraction was collected. Make sure that the column does not dry up.
8. When the sample has entered the gel beads, 50mM ammonium bicarbonate will then be added to the column constantly to fill up the rest of the fraction until all the fraction have 2ml of sample elution.
9. The 8 samples were then taken to test for their absorbance value.

Analysis (Stage 3)

- Spectrophotometry

Absorbance readings were taken using a Spectrophotometer set at 476 nm . ( The wavelength at which GFP is strongly absorbs and gives out its usual florescence).