RFP Lab
Procedure:
Create RFP (red florescence protein) using e-coli bacteria and to learn about genetic engineering
Materials+ procedures:
Lab 2A - materials and procedure can be found in Amgen lab manual 2a
Lab 4A - materials and procedure can be found in Amgen lab manual 4a
Lab 5A - materials and procedure can be found in Amgen lab manual 5a
Lab 6A - materials and procedure can be found in Amgen lab manual 6a
Part 2a- Verification of plasmid by restriction digest:
We used BamH I and Hind III to cut the bacterial plasmid, allowing us to isolate and extract the desired RFP-ara gene
Part 4a: verification of plasmid digest using electrophoresis:
Using electrophoresis, we were able to prove the RFP gene was accepted into the new plasmid
Part 5a: transformation of bacteria with recombinant plasmid.
Restriction enzymes were used to cut our plasmid and insert the RFP gene, making it a recombinant plasmid
Part 6: purification of RFP using chromatography.
Having breached the bacteria membranes, we separated them from the RFP and purified the protein in a chromatography column.
Results:
Lab 2a and 4a:
After running the gel, we found that the size of our molecule corresponded with the DNA ladder, meaning that our DNA molecule was the right one and could be used in the next step. <image not available yet>
Lab 5a:
In order to purify the RFP, the bacteria had to first produce it. We incubated it in a petri dish in order for the bacteria to multiply. In true HS Bio fashion, the bacteria grew and none of it was red.
Lab 6a:
Replacement bacteria was used to create the RFP and they were the ones that were broken down and put in the centrafuge to separate the protein. The protein was then put through a chromatography column and purified.
Conclusion:
This was one of the longer labs we completed this semester and one of the more complicated. Nevertheless, our group worked well together on the project, though it was not always clear what the project was. The multiple sections and processes of this lab made it confusing at times, but eventually, we were able to figure out what was going on. Towards the end, we were able to experiment with the chromatography pump which had not been used before. it did not nescessarily make the task easier, but it did add some change to what would have been a rather monotonous procedure.
Create RFP (red florescence protein) using e-coli bacteria and to learn about genetic engineering
Materials+ procedures:
Lab 2A - materials and procedure can be found in Amgen lab manual 2a
Lab 4A - materials and procedure can be found in Amgen lab manual 4a
Lab 5A - materials and procedure can be found in Amgen lab manual 5a
Lab 6A - materials and procedure can be found in Amgen lab manual 6a
Part 2a- Verification of plasmid by restriction digest:
We used BamH I and Hind III to cut the bacterial plasmid, allowing us to isolate and extract the desired RFP-ara gene
Part 4a: verification of plasmid digest using electrophoresis:
Using electrophoresis, we were able to prove the RFP gene was accepted into the new plasmid
Part 5a: transformation of bacteria with recombinant plasmid.
Restriction enzymes were used to cut our plasmid and insert the RFP gene, making it a recombinant plasmid
Part 6: purification of RFP using chromatography.
Having breached the bacteria membranes, we separated them from the RFP and purified the protein in a chromatography column.
Results:
Lab 2a and 4a:
After running the gel, we found that the size of our molecule corresponded with the DNA ladder, meaning that our DNA molecule was the right one and could be used in the next step. <image not available yet>
Lab 5a:
In order to purify the RFP, the bacteria had to first produce it. We incubated it in a petri dish in order for the bacteria to multiply. In true HS Bio fashion, the bacteria grew and none of it was red.
Lab 6a:
Replacement bacteria was used to create the RFP and they were the ones that were broken down and put in the centrafuge to separate the protein. The protein was then put through a chromatography column and purified.
Conclusion:
This was one of the longer labs we completed this semester and one of the more complicated. Nevertheless, our group worked well together on the project, though it was not always clear what the project was. The multiple sections and processes of this lab made it confusing at times, but eventually, we were able to figure out what was going on. Towards the end, we were able to experiment with the chromatography pump which had not been used before. it did not nescessarily make the task easier, but it did add some change to what would have been a rather monotonous procedure.