Spring 2024: Week 13 - April 22, 2024 - April 26, 2024
Introduction
Our goal this week is to do Gibson Assembly. We have almost all of the fragments that we need to get started except for the left fragment. We do not have enough of our sample so we will need to get more.
Methods
4/22/24
Until we have successfully assembled and done transformation, we will be keeping plates of our cultures in case there is a need to extract from them again. For this reason, we streaked D. radiodurans (D.rad), E. coli pRAD1 and E. coli pRHAM from freezebacs onto plates.
Working under the hood, we inoculated each plate using a four way streak. We inoculated D. rad onto TGY agar plates, E. coli pRAD1 onto LB ampicillin plates, and E. coli pRHAM onto LB kanamycin plates. The D. rad plate was placed into the 30℃ incubator and the two E. coli plates were placed into the 37℃ incubator.
4/23/24
Since the plates we inoculated on 4/23/24 are coming from freezebac, they must be streaked onto another plate before being inoculated into broth. This allows the bacteria to fully wake up and it will give us the most optimal cells. Today, we streaked the E. coli pRAD1 and the E. coli pRHAM from plate onto plate using our most sterile technique.
We went on to make a 1% gel to use later on in the week after we do PCR on our raw left fragment to get a higher yield. To make the gel, we added 30 ml of TAE buffer into a 125 ml flask. To the flask, we added 0.3 g of agarose. We made a cork out of tightly rolled up paper towels and placed the flask in the microwave. We microwaved it until the agarose dissolved completely and it became a clear homogenized solution. The flask cooled for 5 mins before being poured into the tray of the gel electrophoresis apparatus. Since the gel was being made in advance, we let it cool completely and wrapped it up in syran wrap before placing it in the refrigerator.
Since we are preparing to do Gibson Assembly, we nanodropped each of our fragments to see their concentrations. We checked the unclean fragments that are results from extractions. We checked each extraction and picked out the tubes with the best concentrations to use them for assembly.
4/24/24
We performed a 3-way Gibson Assembly using our pRHAM, left and right fragments. We diluted each of the samples in a PCR tube by adding 2 µL of each sample and 2 µL of PCR water. Each sample was diluted separately. They were mixed by pop spinning them. We then mixed all three fragments together in a PCR tube. 2 µL of the left fragment, 3 µL of the right fragment, and 2.5 µL of the pRHAM fragment was added into a tube. We then added 2.5 µL of PCR water to make it 10 µL. To the same tube, we added 10 µL of Gibson mastermix that contained the primers. We then mixed it all together by pop spinning the tube. The tube was placed into the Thermocycler on the “incubate” setting at 50℃ for 1 hour.
Right after the Gibson procedure was done, we took the tube out of the Thermocycler and got it ready for PCR. We took 2 µL of Gibson product and mixed it with 18 µL of PCR mastermix by spinning it in the popspinner. We also added in a control tube which contained 18 µL of PCR mastermix and 2 µL of D. radiodurans genomic extraction. The Thermocycler ran at the following times and temperatures:
95℃ for 4 minutes
95℃ for 30 seconds
60℃ for 30 seconds
72℃ for 30 seconds
Repeats steps 2-4 30 times
72℃ for 7 mins
10℃ hold
4/25/24
To confirm that our Gibson Assembly was successful, we ran a gel. The first well contained the 10 µL of the 1 kb ladder. The second well had the Gibson Assembly product. The third well had the 100 bp ladder. The fourth well had our genomic extraction PCR using the primers made for Gibson. The second and fourth wells had 2 µL of loading dye mixed with 10 µL of sample.
Results
4/23/24
pRHAM
Left Fragment
Right Fragment
pRAD1 (Vector)
4/25/24
Gel electrophoresis results of our Gibson Assembly product.
Discussion
4/24/24
We decided to try using 2 µL of genomic extraction from D. radiodurans with the primers. In theory, the primers should recognize the DNA and prime to it. If it doesn’t then we have reason to believe that there is a problem with the primers. The primers have not been tested before so this will serve as a control.
When the PCR reaction was done, we ran out of time to run a gel so we placed it in the -20℃ freezer. We clearly labeled which tube contained our Gibson reaction and which one contained our genomic DNA.
4/25/24
Our Gibson Assemply product had a band where we expected to see one if Gibson was successful. We expected to have a fragment of about 2,527 bp. We had a faint band about that size in the well with our Gibson product. Interestingly enough, the well with our genomic extraction did not have a band. This is odd because this well was supposed to serve as our control so that we would have something to compare the Gibson product to.
Conclusion
We have had a successful week so far. Our Gibson seems to be successful. Since the band that we got is faint, we plan on running it through PCR againin hopes of amplifying our sample. Amplifying our sample will hopefully give us a brighter band and help us confirm that our assembly was successful.
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