Spring 2024: Week 3 - February 5, 2024- February 9, 2024
Introduction:
After getting unwanted results during last week’s PCR cleanup, we needed to try another round of cleanup on our duplicate samples to see what our next steps would be. If the second PCR cleanup goes well, we can move forward with Gibson Assembly. If it shows similar results to last week’s, we will have to go back to the extraction process.
Methods:
2/5/2024
We did our second PCR product cleanup on our duplicate samples for our Left Fragment and our pRHAM plasmid extraction. We followed the same procedure as last Thursday but we made a small change. Instead of adding 6 μL of DNA Elution Buffer, we added 10 μL.
2/7/2024
We were not able to get our materials to do the pRHAM plasmid extractions so we pushed it back a day. Instead, we were able to make freezebacs of our E.coli pRHAM culture. We made three freezebacs and put them in the -80°C freezer for long term storage.
As housekeeping, we inoculated D. radiodurans from one of our plates into TGY broth. Until we are able to confirm that we have everything that we need for assembly, we are going to be keeping continuous cultures of each of the bacteria that we are working with. We will be inoculating into new, sterile broth every three days.
2/8/2024
We pulled out our D. radiodurans genomic extraction products from the -80°C freezer and put it on ice. Since we still had sufficient volume in each of our genomic extraction tubes, we did not have to perform a new genomic extraction like we had originally planned. We mixed 18 μL of mastermix with 2 μL of our sample. We had 4 tubes in the freezer and we ran PCR on each of them. We did four tubes in total. The ThermoCycler ran at the following temperatures and times:
95°C for 4 minutes
95°C for 30 seconds
60°C for 30 seconds
72°C for 30 seconds
Repeat steps 2-4 (30x)
72°C for 7 minutes
10°C for ∞ (this is a hold)
While we had the ThermoCycler running, we did a plasmid extraction on E.coli pRHAM. We used the Zyppy Plasmid Miniprep Kit and followed the protocol. Since it has given us our most optimal results in the past, we compacted the cells once before beginning. This consists of adding 1 mL of bacterial culture, spinning it in the centrifuge for 1 minute, and discarding the supernatant. We then begin the protocol with step 1.
Results:
2/5/2024
PCR Product Cleanup
Nanodrop results of our second PCR product cleanup of our Left Fragment and our pRHAM.
2/8/2024
E.coli pRHAM Plasmid Extraction
Nanodrop results of pRHAM plasmid extraction.
D.rad PCR
Nanodrop results of our D.rad genomic extraction PCR
Conclusion:
2/5/2024
Our nanodrop values were still too low after our second PCR cleanup. We could not use them for assembly. We had to do another extraction to get the pRHAM plasmid out of our E.coli sample.
2/8/2024
The nanodrop results for our plasmid extraction were not what we expected and we will not be able to use these samples either. On Monday, we will run it on a gel to see if the plasmid show up. If it does, we have been given the green light to do PCR and if not, we will perform another extraction.
A reason why the values might be so low could be because our culture is older than what we should be using for extraction. This culture had been inoculated on 2/5/2024 and we used it for extraction on 2/8/2024. Since E.coli grows so quickly, the broth is considered old. This plasmid also has a kanamycin resistance so we add in kanamycin when we inoculate. Perhaps, by the time we use it for extraction, the bacteria has already broken down all of the kanamycin we have provided and is beginning to expel the plasmid itself. While nothing is confirmed, these are only our thoughts as of now. From now on when working with E.coli, we will inoculate, let it grow for 24 hours, then use it.
As for the D. rad PCR results, we had excellent results. The nanodrop results were exactly what we were looking for. We will be able to use these samples moving forwards. Next week, we will clean them up using the same PCR Product Cleanup procedure that we used on Monday where we added 10 μL of DNA Elution Buffer.
Introduction:
After getting unwanted results during last week’s PCR cleanup, we needed to try another round of cleanup on our duplicate samples to see what our next steps would be. If the second PCR cleanup goes well, we can move forward with Gibson Assembly. If it shows similar results to last week’s, we will have to go back to the extraction process.
Methods:
2/5/2024
We did our second PCR product cleanup on our duplicate samples for our Left Fragment and our pRHAM plasmid extraction. We followed the same procedure as last Thursday but we made a small change. Instead of adding 6 μL of DNA Elution Buffer, we added 10 μL.
2/7/2024
We were not able to get our materials to do the pRHAM plasmid extractions so we pushed it back a day. Instead, we were able to make freezebacs of our E.coli pRHAM culture. We made three freezebacs and put them in the -80°C freezer for long term storage.
As housekeeping, we inoculated D. radiodurans from one of our plates into TGY broth. Until we are able to confirm that we have everything that we need for assembly, we are going to be keeping continuous cultures of each of the bacteria that we are working with. We will be inoculating into new, sterile broth every three days.
2/8/2024
We pulled out our D. radiodurans genomic extraction products from the -80°C freezer and put it on ice. Since we still had sufficient volume in each of our genomic extraction tubes, we did not have to perform a new genomic extraction like we had originally planned. We mixed 18 μL of mastermix with 2 μL of our sample. We had 4 tubes in the freezer and we ran PCR on each of them. We did four tubes in total. The ThermoCycler ran at the following temperatures and times:
95°C for 4 minutes
95°C for 30 seconds
60°C for 30 seconds
72°C for 30 seconds
Repeat steps 2-4 (30x)
72°C for 7 minutes
10°C for ∞ (this is a hold)
While we had the ThermoCycler running, we did a plasmid extraction on E.coli pRHAM. We used the Zyppy Plasmid Miniprep Kit and followed the protocol. Since it has given us our most optimal results in the past, we compacted the cells once before beginning. This consists of adding 1 mL of bacterial culture, spinning it in the centrifuge for 1 minute, and discarding the supernatant. We then begin the protocol with step 1.
Results:
2/5/2024
PCR Product Cleanup
Nanodrop results of our second PCR product cleanup of our Left Fragment and our pRHAM.
2/8/2024
E.coli pRHAM Plasmid Extraction
Nanodrop results of pRHAM plasmid extraction.
D.rad PCR
Nanodrop results of our D.rad genomic extraction PCR
Conclusion:
2/5/2024
Our nanodrop values were still too low after our second PCR cleanup. We could not use them for assembly. We had to do another extraction to get the pRHAM plasmid out of our E.coli sample.
2/8/2024
The nanodrop results for our plasmid extraction were not what we expected and we will not be able to use these samples either. On Monday, we will run it on a gel to see if the plasmid show up. If it does, we have been given the green light to do PCR and if not, we will perform another extraction.
A reason why the values might be so low could be because our culture is older than what we should be using for extraction. This culture had been inoculated on 2/5/2024 and we used it for extraction on 2/8/2024. Since E.coli grows so quickly, the broth is considered old. This plasmid also has a kanamycin resistance so we add in kanamycin when we inoculate. Perhaps, by the time we use it for extraction, the bacteria has already broken down all of the kanamycin we have provided and is beginning to expel the plasmid itself. While nothing is confirmed, these are only our thoughts as of now. From now on when working with E.coli, we will inoculate, let it grow for 24 hours, then use it.
As for the D. rad PCR results, we had excellent results. The nanodrop results were exactly what we were looking for. We will be able to use these samples moving forwards. Next week, we will clean them up using the same PCR Product Cleanup procedure that we used on Monday where we added 10 μL of DNA Elution Buffer.
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