Day 5 in BPT lab

Hey hey...

Yea... Day5... We did Isolation and Purification of the Product... For me, I personally like this part of the whole experiment coz.... I dun really know why... hahaha... Just kinda like it... weehee... :P

Ok... let me share with u guys da procedure for Isolation and purification... *I wrote the procedures in proper sentences coz wouldnt wan u guys out there to get confused* hehe...

So here it is:

Procedure of the isolation and purification of GF

Stage 1: Isolation of GFP

There are 3 methods for Isolation of GFP; Usage of Enzymes, Freezing and Thawing, and Sonication

1. Usage of Enzymes: By using micropipettor, the pellet in 500 µl of TE buffer of pH 7.5 being resuspended till no formation of visible clumps. 2 drops of lysozyme being added to the resuspended cell pellet using the disposable 1 ml plastic pipette where it will initiate enzymatic digestion of the bacteria cell wall. The ezymes are allow to act for 15 mins

2. Freezing and Thawing: Tube being placed in liquid nitrogen until the contents are frozen where later, warm water was used to thaw it. To complete the rupturing of the bacteria cell wall, the freezing and thawing being done twice. The reason why freezing and thawing being carried out is to add mechanical stress to the cell wall as the cell water content expands and contracts while being frozen and thawed respectively.

3. Sonication: In completion of cell disruption, process of sonication being carried out where the ultrasonic waves cause the bacteria cell wall to implode under the vibrational pressure. Sonication is done on ice for 4 cycles of 25 seconds rest in between sonication cycles. The content were then being centrifuged for 20 minutes at 10 000 rpm after cell disruption. The pellet and supernatant being separated out where the pellet being suspended using 400 µl of TE buffer. The product of GFP is the supernatant where it can be determined by observing under UV light. (In carrying out this whole Sonication method, protective ear muffs being worn at all times).

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Stage 2: Purification

1. 8 Test tubes were labelled from 1 to 8, and a Blank


2. Test tube labelled Blank bening filled with 2 ml of ammonium bicarbonate. This Blank being used as a guide to mark the rest of the test tubes with a line at the 2 ml level.


3. Column was carefully drained into a waste beaker until the buffer was just even with the top of the gel bed.


4. Cell-free extract were transferred using glass pipette to the top of the gel bed by gently swirling the pipette around inside the edge of column, just above the packed matrix.


5. Fractions were taken by removing the waste beaker and placing a test tune under the stopcock. On this point of time, eluant was collected in test tubes where each test tube were filled to the 2 ml mark.


6. Slowly open the stopcock, sample were allowed to flow completely into the gel bed where eluting buffer were collected in the first tube. Flow rate were adjusted to a 1drop per 2 sec interval. The entire column were not left to dry out.


7. 50 mM ammonium bicarbonate were added t the top of the column while taking the fractions. To provide consistent flow of buffer through the chromatography matrix, 3 cm column of buffer were maintained on top of the gel.


8. Step 7 were repeated till the 8^th tube is filled.

Weeheee... hope dat da procedure mentioned above is clear enough... hehe... Adios :P