RFP Lab
Purpose: Make RFP from jelly fish in bacteria and to learn about the steps of genetic engineering
Materials/Procedure:
2a- materials and procedure can be found in Amgen lab manual part 2a
4a- materials and procedure can be found in Amgen lab manual part 4a
5a- materials and procedure can be found in Amgen lab manual part 5a
6a- materials and procedure can be found in Amgen lab manual part 6a
Experimental Overview:
Part 2a: Verification of plasmid by restriction digest. We cut the plasmid with BamH1 and Hind III to cut out RFP-ara from bacteria plasmid.
Part 4a: Verification of plasmid digest by electrophonesis. We used the process of electrophoresis to ensure that we had actually cut the plasmid correctly.
Part 5a: Transformation of bacteria with recombinant plasmid. We changed the bacteria using a recombinant plasmid. Using resrtiction enzymes, we took the gene we wanted and put it with the bacteria.
Part 6a: Verification RFPusing chromatography. We used chromatography to separate the RFP from everything else.
Lab2a
1. If we cut the plasmid and the insulin gene from the DNA so the ends of them connect and fit together.
Why does using two different enzymes to cut the plasmid prevent the plasmid from reforming a circle without the
inserted gene?
2. 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.
3.So we can keep the lab controlled and it is important because we can base our answers off it.
4.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
Lab 5a
5a Questions
Lab 6a
Final step:
Overview: Now that we have collected our RFP protein from the bacteria, there is one more thing to do. We need to run a gel, only this time with the RFP protein. We ran the protein next to a Protein ladder to check if it was the right size. And if there is only single bands it means the protein is pure because there is only one type of protein.
Analysis/Conclusion
Our red conies didn't show up so our experiment was not fully complete, but this was the same for most of the other groups as well.
Reflection
I enjoyed working with different people in my group becasue it gave us a chance to collaborate with others, not just our freinds who we usually work with. I learned quite a lot throughout these labs but the only problem I had was the challenge of difficulty this lab had compared to previous labs, which is a big factor to why I enjoyed more than the other labs. Overall I enjoyed it and learned a lot.
Materials/Procedure:
2a- materials and procedure can be found in Amgen lab manual part 2a
4a- materials and procedure can be found in Amgen lab manual part 4a
5a- materials and procedure can be found in Amgen lab manual part 5a
6a- materials and procedure can be found in Amgen lab manual part 6a
Experimental Overview:
Part 2a: Verification of plasmid by restriction digest. We cut the plasmid with BamH1 and Hind III to cut out RFP-ara from bacteria plasmid.
Part 4a: Verification of plasmid digest by electrophonesis. We used the process of electrophoresis to ensure that we had actually cut the plasmid correctly.
Part 5a: Transformation of bacteria with recombinant plasmid. We changed the bacteria using a recombinant plasmid. Using resrtiction enzymes, we took the gene we wanted and put it with the bacteria.
Part 6a: Verification RFPusing chromatography. We used chromatography to separate the RFP from everything else.
Lab2a
1. If we cut the plasmid and the insulin gene from the DNA so the ends of them connect and fit together.
Why does using two different enzymes to cut the plasmid prevent the plasmid from reforming a circle without the
inserted gene?
2. 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.
3.So we can keep the lab controlled and it is important because we can base our answers off it.
4.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
- If you don't have the correct recombinant plasmid the damaged bacteria will go unnoticed because it might not be visible, therefore you would have to restart the lab.
- My gel results were the same as my predictions because they had the same number of bands in each lane as I had predicted.
- There is a couple of bands unexpected and this could be a result of either human error or we damaged the gel with the DNA.
- Yes because we got the correct results and the R+ lanes had two fragments, which was desired.
- Yes, I saw multiple different bands showing that the plasmid was digested.
- Yes, there were multiple bands showing the plasmid was digested into multiple fragments.
- The RFP gene would be found in the R+ lane and the ampR gene in R- lane.We can locate these in both of these lanes.
- The lanes with plasmids are supercoiled and both migrate. Yes the fragments have elongated edges while the plasmids are supercoiled.
Lab 5a
- The P+ was given a plasmid while P- wasn't. The P- bacteria culture is meant to become a plasmid controller.
- They need to recuperate and express its antibiotic resistance.
- To create an environment to have the ability to grow like cell division
- So we can control the experiment and keep the substance from being contaminated.
5a Questions
- Our predictions were correct but we might have accidentally cross contaminated, but doubtful.
- We didn't have any
- They didn't appear.
- Most of the time these are damaged, but if you have a lot we have a better chance of getting the correct answer.
- RFP glows in the gel and protein.
- They have similar compositions as us in terms of structure or organelles.
Lab 6a
- In binding spaces proteins attach to other molecules and this allows it to carry out its function.
- The sequence of the amino acids relates to folding.
- The Elute is brighter than the lysate. The RFP will account for that.
- Hydrophobic amino acids are used as the basis for separation by column chromatography
- Use more of the original buffer to increase the purity of the red fluorescent protein sample.
Final step:
Overview: Now that we have collected our RFP protein from the bacteria, there is one more thing to do. We need to run a gel, only this time with the RFP protein. We ran the protein next to a Protein ladder to check if it was the right size. And if there is only single bands it means the protein is pure because there is only one type of protein.
Analysis/Conclusion
Our red conies didn't show up so our experiment was not fully complete, but this was the same for most of the other groups as well.
Reflection
I enjoyed working with different people in my group becasue it gave us a chance to collaborate with others, not just our freinds who we usually work with. I learned quite a lot throughout these labs but the only problem I had was the challenge of difficulty this lab had compared to previous labs, which is a big factor to why I enjoyed more than the other labs. Overall I enjoyed it and learned a lot.