Team:Victoria Australia/Project/Materials and Methods

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== <div style="width: 60%; height: 60%; color: #50cad; font-size: 25px; ">Materials & Methods</div> ==
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To begin with, reagent solutions were made up. LB Agar plates (protocol 1) were made using 10g tryptone digest of casein, 5g yeast extract, 5g NaCl, 15g agar and then made up to 1L using milliQ water. Ampicillin (100g/ml) was then prepared by adding 1gram of ampicillin and 9 ml (9gram) milliQ water so that the total volume was 10ml. LB media was made using 10g tryptone, 5g yeast extract, 5g NaCl and milliQ water to make the media up to 1L.  
To begin with, reagent solutions were made up. LB Agar plates (protocol 1) were made using 10g tryptone digest of casein, 5g yeast extract, 5g NaCl, 15g agar and then made up to 1L using milliQ water. Ampicillin (100g/ml) was then prepared by adding 1gram of ampicillin and 9 ml (9gram) milliQ water so that the total volume was 10ml. LB media was made using 10g tryptone, 5g yeast extract, 5g NaCl and milliQ water to make the media up to 1L.  
The bacterial transformation of both BFP and GFP was completed by firstly Pre-incubating the LB-agar plates at 37°C for 2 hours prior. The Ca-competent cells were thawed on ice (4ºC).  Plasmid DNA was added and the tubes stored on ice for 30mins. The tubes were transferred to a rack placed in a preheated water bath (45ºC) and incubated for 30secs. The tubes were then transferred to ice (4ºC) and incubated on ice for 2 min. 800μl of SOC medium was added to each tube and Incubated at 37ºC with gentle shaking (no more that 50rpm) for 45min. 50μl of the transformed cells was transferred onto the LB agar plates each containing ampicillin, and spread the cell suspension thoroughly on the surface using a sterile rod (Streaking). Plates were incubated (agar side up) at 37ºC for ~16h.  
The bacterial transformation of both BFP and GFP was completed by firstly Pre-incubating the LB-agar plates at 37°C for 2 hours prior. The Ca-competent cells were thawed on ice (4ºC).  Plasmid DNA was added and the tubes stored on ice for 30mins. The tubes were transferred to a rack placed in a preheated water bath (45ºC) and incubated for 30secs. The tubes were then transferred to ice (4ºC) and incubated on ice for 2 min. 800μl of SOC medium was added to each tube and Incubated at 37ºC with gentle shaking (no more that 50rpm) for 45min. 50μl of the transformed cells was transferred onto the LB agar plates each containing ampicillin, and spread the cell suspension thoroughly on the surface using a sterile rod (Streaking). Plates were incubated (agar side up) at 37ºC for ~16h.  
Following the transformation, protein expression was carried out by adding 100ml of Lb media into two conical flasks that were previously autoclaved. Using a pipette tip a single colony from the transformed bacteria of GFP and BFP was selected and dropped into conical flask. They were then incubated at 240rpm at 38 ºC for 18 hours.
Following the transformation, protein expression was carried out by adding 100ml of Lb media into two conical flasks that were previously autoclaved. Using a pipette tip a single colony from the transformed bacteria of GFP and BFP was selected and dropped into conical flask. They were then incubated at 240rpm at 38 ºC for 18 hours.
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Using the incubated GFP and BFP they were divided into two falcon tubes each and centrifuged at 3000rpm for  
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Using the incubated GFP and BFP they were divided into two falcon tubes each and centrifuged at 3000rpm for 20min. The supernatant was emptied (waste) and the pellet was resuspended with 20ml of SOC solution and 20 µL of ampicillin being added. At a wavelength of 600nm, the solution was measured in the spectrometer to check the density of the bacteria. The results obtained were as follows: GFP = 0.93A and BFP = 0.95A.  
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20min. The supernatant was emptied (waste) and the pellet was resuspended with 20ml of SOC solution and 20 µL of ampicillin being added. At a wavelength of 600nm, the solution was measured in the spectrometer to check the density of the bacteria. The results obtained were as follows: GFP = 0.93A and BFP = 0.95A.  
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Two conical flasks were made up with 120ml SOC solution and 120 µL of Ampicillin (this is the solution in which GFP and BFP are added to). Using C1V1=C2V2 the amount of GFP and BFP that was added to the solution from above (SOC and Ampicillin) was found. After adding the GFP/BFP the solution was incubated at 240rpm at 28 ºC for 1 hour. Following this hour the absorbance was read at 600nm and the following results were obtained: GFP = 0.7A (undiluted) and BFP = 0.64A (undiluted).  
Two conical flasks were made up with 120ml SOC solution and 120 µL of Ampicillin (this is the solution in which GFP and BFP are added to). Using C1V1=C2V2 the amount of GFP and BFP that was added to the solution from above (SOC and Ampicillin) was found. After adding the GFP/BFP the solution was incubated at 240rpm at 28 ºC for 1 hour. Following this hour the absorbance was read at 600nm and the following results were obtained: GFP = 0.7A (undiluted) and BFP = 0.64A (undiluted).  
Transformation of Vic GFP, BFP, YFP, AZ, blueberry and cherry was done using the same protocol as above.  
Transformation of Vic GFP, BFP, YFP, AZ, blueberry and cherry was done using the same protocol as above.  
Following these transformations, more reagents were prepared. Phosphate buffer solution was made by adding 1 tablet of phosphate buffered saline to 100ml milliQ water. Chaps had also been made using 0.017g chaps in 1ml of milliQ water. Lysis Buffer 1 was created using 250 µL chaps, 500 µL sucrose and made up to 10ml using the phosphate buffer. 100ml milliQ water was added to 34.2g sucrose making 1M sucrose solution.  1M imieazole was prepared by adding 0.6808g of imieazole to 10ml of milliQ water.
Following these transformations, more reagents were prepared. Phosphate buffer solution was made by adding 1 tablet of phosphate buffered saline to 100ml milliQ water. Chaps had also been made using 0.017g chaps in 1ml of milliQ water. Lysis Buffer 1 was created using 250 µL chaps, 500 µL sucrose and made up to 10ml using the phosphate buffer. 100ml milliQ water was added to 34.2g sucrose making 1M sucrose solution.  1M imieazole was prepared by adding 0.6808g of imieazole to 10ml of milliQ water.
1ml lysis buffer was then added to pellets containing either GFP/BFP in order to resuspend the pellet. After resuspending, a Pasteur pipette was used to remove 1ml into another pellet also containing GFP or BFP. That is; GFP with lysis buffer added to pellet
1ml lysis buffer was then added to pellets containing either GFP/BFP in order to resuspend the pellet. After resuspending, a Pasteur pipette was used to remove 1ml into another pellet also containing GFP or BFP. That is; GFP with lysis buffer added to pellet
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Revision as of 01:56, 19 October 2009

Finalised fluoroforce header.jpg




Materials & Methods


Contents

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To begin with, reagent solutions were made up. LB Agar plates (protocol 1) were made using 10g tryptone digest of casein, 5g yeast extract, 5g NaCl, 15g agar and then made up to 1L using milliQ water. Ampicillin (100g/ml) was then prepared by adding 1gram of ampicillin and 9 ml (9gram) milliQ water so that the total volume was 10ml. LB media was made using 10g tryptone, 5g yeast extract, 5g NaCl and milliQ water to make the media up to 1L. The bacterial transformation of both BFP and GFP was completed by firstly Pre-incubating the LB-agar plates at 37°C for 2 hours prior. The Ca-competent cells were thawed on ice (4ºC). Plasmid DNA was added and the tubes stored on ice for 30mins. The tubes were transferred to a rack placed in a preheated water bath (45ºC) and incubated for 30secs. The tubes were then transferred to ice (4ºC) and incubated on ice for 2 min. 800μl of SOC medium was added to each tube and Incubated at 37ºC with gentle shaking (no more that 50rpm) for 45min. 50μl of the transformed cells was transferred onto the LB agar plates each containing ampicillin, and spread the cell suspension thoroughly on the surface using a sterile rod (Streaking). Plates were incubated (agar side up) at 37ºC for ~16h. Following the transformation, protein expression was carried out by adding 100ml of Lb media into two conical flasks that were previously autoclaved. Using a pipette tip a single colony from the transformed bacteria of GFP and BFP was selected and dropped into conical flask. They were then incubated at 240rpm at 38 ºC for 18 hours. Using the incubated GFP and BFP they were divided into two falcon tubes each and centrifuged at 3000rpm for 20min. The supernatant was emptied (waste) and the pellet was resuspended with 20ml of SOC solution and 20 µL of ampicillin being added. At a wavelength of 600nm, the solution was measured in the spectrometer to check the density of the bacteria. The results obtained were as follows: GFP = 0.93A and BFP = 0.95A. Two conical flasks were made up with 120ml SOC solution and 120 µL of Ampicillin (this is the solution in which GFP and BFP are added to). Using C1V1=C2V2 the amount of GFP and BFP that was added to the solution from above (SOC and Ampicillin) was found. After adding the GFP/BFP the solution was incubated at 240rpm at 28 ºC for 1 hour. Following this hour the absorbance was read at 600nm and the following results were obtained: GFP = 0.7A (undiluted) and BFP = 0.64A (undiluted). Transformation of Vic GFP, BFP, YFP, AZ, blueberry and cherry was done using the same protocol as above. Following these transformations, more reagents were prepared. Phosphate buffer solution was made by adding 1 tablet of phosphate buffered saline to 100ml milliQ water. Chaps had also been made using 0.017g chaps in 1ml of milliQ water. Lysis Buffer 1 was created using 250 µL chaps, 500 µL sucrose and made up to 10ml using the phosphate buffer. 100ml milliQ water was added to 34.2g sucrose making 1M sucrose solution. 1M imieazole was prepared by adding 0.6808g of imieazole to 10ml of milliQ water. 1ml lysis buffer was then added to pellets containing either GFP/BFP in order to resuspend the pellet. After resuspending, a Pasteur pipette was used to remove 1ml into another pellet also containing GFP or BFP. That is; GFP with lysis buffer added to pellet


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