RFP Lab - 12/16/14
Purpose - Use RFP from jelly fish in bacteria and learn about it with 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
-Cut a plasmid with BanHI + HindIII to cut out RFP-ara gene from the bacteria plasmid
Part 4a - Verifcation of plasmid digest by electophoresis
-Used electrophoresis and DNA ladder too sequence the digest
Part 5a - Transformation of bacteria with recombinant plasmid
-Make bacteria into recombinant plasmid with restrictions
Part 6a - Purification of RFP using chromatography
-Here we isolated the RFP
Data -
2a - 1. ori-origin of replication, GoI-gene of interest, Amp-R-selective marker, Ara-C-binds to the promoter so we can get transcription of our GoI. 2. restriction enzymes in nature are defense mechanisms that cut DNA from other bacteria. 3. Bacteria retains an antibiotic resistance because otherwise they would die off. In the medical field they have to create a whole new class of antibiotics. 4. Bacteria have DNA, RNA polymerase, and ribosomes (central dogma) just like humans. We can replace DNA with other DNA that can change a specific trait.
4a - 1. Since the recombinant plasmid has the desired trait you want to make sure that it really has the desired trait. 2. The gel results compared different to our comparisons because the DNA ladder was not visible due to a malfunction somewhere. 3. No I don't believe so, besides the opposite of not showing up. 4. Well without the DNA ladder we can't conclude this question as for evidence because we cannot estimate the measures. 5. No just one band. 6. RFP to R+ and AMP R to R-.
5a - 1. My predictions were correct but we did miss one of our pipeting instances where we crossed the middle line. 2. None. 3. They did not appear. 4. because not a lot is used as desirable, the more the better. 5. RFP gives the red flourescent glow to our proteins. 6. bacteria can make human protein because they have like organelles and cell processes.
Analysis/conclusion - In the end, our red flourescent protein was not visible, it turns out that the gene we received did not work properly so our end result wasn't very optimistic.
Reflection - My group was consisted of good work ethic people and we collaborated nicely. I thought the lab was really drawn out and long as which we skipped around a bit. The most challenging task I had was gripping the content of the lab. I found that the more I didn't know the worse off I was, although I did learn much of the basics which were interesting. In the end it definitely wasn't my favorite but was rather the most challenging and interesting.
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
-Cut a plasmid with BanHI + HindIII to cut out RFP-ara gene from the bacteria plasmid
Part 4a - Verifcation of plasmid digest by electophoresis
-Used electrophoresis and DNA ladder too sequence the digest
Part 5a - Transformation of bacteria with recombinant plasmid
-Make bacteria into recombinant plasmid with restrictions
Part 6a - Purification of RFP using chromatography
-Here we isolated the RFP
Data -
2a - 1. ori-origin of replication, GoI-gene of interest, Amp-R-selective marker, Ara-C-binds to the promoter so we can get transcription of our GoI. 2. restriction enzymes in nature are defense mechanisms that cut DNA from other bacteria. 3. Bacteria retains an antibiotic resistance because otherwise they would die off. In the medical field they have to create a whole new class of antibiotics. 4. Bacteria have DNA, RNA polymerase, and ribosomes (central dogma) just like humans. We can replace DNA with other DNA that can change a specific trait.
4a - 1. Since the recombinant plasmid has the desired trait you want to make sure that it really has the desired trait. 2. The gel results compared different to our comparisons because the DNA ladder was not visible due to a malfunction somewhere. 3. No I don't believe so, besides the opposite of not showing up. 4. Well without the DNA ladder we can't conclude this question as for evidence because we cannot estimate the measures. 5. No just one band. 6. RFP to R+ and AMP R to R-.
5a - 1. My predictions were correct but we did miss one of our pipeting instances where we crossed the middle line. 2. None. 3. They did not appear. 4. because not a lot is used as desirable, the more the better. 5. RFP gives the red flourescent glow to our proteins. 6. bacteria can make human protein because they have like organelles and cell processes.
Analysis/conclusion - In the end, our red flourescent protein was not visible, it turns out that the gene we received did not work properly so our end result wasn't very optimistic.
Reflection - My group was consisted of good work ethic people and we collaborated nicely. I thought the lab was really drawn out and long as which we skipped around a bit. The most challenging task I had was gripping the content of the lab. I found that the more I didn't know the worse off I was, although I did learn much of the basics which were interesting. In the end it definitely wasn't my favorite but was rather the most challenging and interesting.