RFP lab
Purpose
Make RFP from jelly fish in bacteria
Learn about steps genetic engineering
Make RFP from jelly fish in bacteria
Learn about steps genetic engineering
Materials + Procedure
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 6A - materials and procedure can be found in Amgen lab manual 6a
Experimental overview
Lab 2A: Verification of Plasmid by Restriction Digest
- In this part of the lab, we had to cut a plasmid with BamH1 and HindIII. We were then able to extract the segment of the RFP-Ara gene.
Lab 4A: Verification of Plasmid Digest by Electrophoresis
- To ensure that we had actually cut the plasmid correctly, we used the process of electrophoresis. This means that we ran the plasmid in a gel alongside a DNA ladder. A DNA ladder has different-sized molecules already programmed into it, so after being run, it provides a "ladder" of bands that can be corresponded with the other lanes in order to decipher their molecule sizes. Because we already knew the size of the RFP gene and the plasmid, we were able to say if the digest had gone smoothly.
Lab 5A: Transformation of Bacteria with Recombinant Plasmid
- The heart of the RFP lab was in this section, where we transformed the bacteria into a recombinant (man-made) plasmid. We used restriction enzymes to cut the plasmid and ligase to paste the gene of interest. We then used a selective marker (Amp-R, resistance to Ampicillin).
Lab 6A: Separating the RFP Gene with Column Chromatography
- After incubating the RFP gene, we used a chromatography column in order to separate the RFP from everything else. We did this by using a column with hydrophilic beads: this effectively collected the RFP in the resin bed. Then we eluted the RFP and knocked it off those beads by using a buffer.
Data
2a- Why is it important that the same enzyme or
enzymes be used to cut both the plasmid and the insulin gene from the
human DNA?
If we cut the plasmid and the insulin gene from the DNA so the ends of them connect and fit together like puzzle pieces.
Why does using two different enzymes to cut the
plasmid prevent the plasmid from reforming a circle without the
inserted gene?
Cutting a plasmid with two different enzymes prevents the plasmid from reforming a circle because the circle can't reform if the two different sides don't fit together. If it was only one enzyme the two sides will just fit together eventually.
In this step, you are asked to set up a tube without
the restriction enzymes, BamHI and HindIII. What is the purpose of this
step, and why is it important?
So we can keep the lab controlled and it is important because we can base our answers off it.
Why might the enzymes work best at 37°C? Why
should the enzymes then be placed in the freezer? (Hint: The human body
temperature is 37°C.)
It can grow the rate as if it was in your body and we would put the enzymes in the freezer so they wouldn't grow any more.
lab 4a
The DNA is not visible as it moves through the
gel. The loading dye contains the three dyes that you separated in
Laboratory 1.2. Why is it useful to use the loading dye in this lab?
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 6A - materials and procedure can be found in Amgen lab manual 6a
Experimental overview
Lab 2A: Verification of Plasmid by Restriction Digest
- In this part of the lab, we had to cut a plasmid with BamH1 and HindIII. We were then able to extract the segment of the RFP-Ara gene.
Lab 4A: Verification of Plasmid Digest by Electrophoresis
- To ensure that we had actually cut the plasmid correctly, we used the process of electrophoresis. This means that we ran the plasmid in a gel alongside a DNA ladder. A DNA ladder has different-sized molecules already programmed into it, so after being run, it provides a "ladder" of bands that can be corresponded with the other lanes in order to decipher their molecule sizes. Because we already knew the size of the RFP gene and the plasmid, we were able to say if the digest had gone smoothly.
Lab 5A: Transformation of Bacteria with Recombinant Plasmid
- The heart of the RFP lab was in this section, where we transformed the bacteria into a recombinant (man-made) plasmid. We used restriction enzymes to cut the plasmid and ligase to paste the gene of interest. We then used a selective marker (Amp-R, resistance to Ampicillin).
Lab 6A: Separating the RFP Gene with Column Chromatography
- After incubating the RFP gene, we used a chromatography column in order to separate the RFP from everything else. We did this by using a column with hydrophilic beads: this effectively collected the RFP in the resin bed. Then we eluted the RFP and knocked it off those beads by using a buffer.
Data
2a- Why is it important that the same enzyme or
enzymes be used to cut both the plasmid and the insulin gene from the
human DNA?
If we cut the plasmid and the insulin gene from the DNA so the ends of them connect and fit together like puzzle pieces.
Why does using two different enzymes to cut the
plasmid prevent the plasmid from reforming a circle without the
inserted gene?
Cutting a plasmid with two different enzymes prevents the plasmid from reforming a circle because the circle can't reform if the two different sides don't fit together. If it was only one enzyme the two sides will just fit together eventually.
In this step, you are asked to set up a tube without
the restriction enzymes, BamHI and HindIII. What is the purpose of this
step, and why is it important?
So we can keep the lab controlled and it is important because we can base our answers off it.
Why might the enzymes work best at 37°C? Why
should the enzymes then be placed in the freezer? (Hint: The human body
temperature is 37°C.)
It can grow the rate as if it was in your body and we would put the enzymes in the freezer so they wouldn't grow any more.
lab 4a
The DNA is not visible as it moves through the
gel. The loading dye contains the three dyes that you separated in
Laboratory 1.2. Why is it useful to use the loading dye in this lab?
amgen_sg_sequence2_rev2014.pdf | |
File Size: | 5087 kb |
File Type: |