Spring 2024 - Week 7: March 4, 2024 - March 8, 2024

 Introduction 

Last week, we were able to clean up our left fragment and get it into the -20℃ freezer. It was the last fragment that we needed to clean in order to start assembling them together. The goal for this week is to do Gibson Assembly. 

Methods 

3/5/2024 

I started off the week by making more media so that we could use it later on in the week or after spring break. Since our media would be going bad during spring break, I wanted to make new ones to use after assembly. I wouldn’t want older media to cause us to have unwanted results. I made 100 ml of TGY broth, 100 ml of LB broth, and 200 ml of LB + agar. 

We also planned out how we will be plating the bacteria after assembly. We decided on having 8 plates total that will include a negative control, positive control, and two sets of experimentals. We will be doing four plates with no antibiotic and four plates with ampicillin. 

3/6/2024

We poured plates to have them ready for after assembly. We poured 4 with ampicillin and four without. To add the ampicillin, we heated up the  LB + agar, let it cool until it passed the baby bottle test, and added 200 µL of ampicillin. 

3/7/2024

We were able to get the last of our calculations figured out and confirmed. We were set to begin the Gibson Assembly but first, we were asked to check our samples on the fluorometer and the readings did not match up with our calculations. 

Once we were able to figure out the calculations, we began diluting our fragments to the required volume and checking it on the fluorometer. The results that we were getting on the fluorometer were not consistent with our calculations. We decided to check the same diluted samples on the nanodrop to ensure that the fluorometer readings were accurate. We also decided to check our stock sample (clean PCR product) on the nanodrop to make sure that the values that we plugged into our calculations were accurate. 

Results 

3/7/2024

The calculations for the total volume that we will use of each fragment is:

Vector (pRAD1): 38.3 ng/µL x 6 = 229.8 ng  = 0.053 pmol = 6 µL

pRHAM: 62.7 ng/µL = 32/62 ng = 0.053 pmol = 1 µL

Left: 82.4 ng/µL = 25.49 ng =0.053 pmol = 1 µL

Right:  24.7 ng/µL = 28.94 ng = 0.053 pmol = 1.172 µL

Total pmol: 0.212 pmol


Left Fragment Fluorometer Reading

Sample 

ng/µL

Left Fragment 

7.42

  • 1 µL L. F.

8.80


Left Fragment Clean PCR Product Diluted

Sample 

ng/µL

A260/A280

A260/A230

Left Fragment 

39.9

1.89

0.96


Stock Sample Nanodrop Readings 


Sample 

ng/µL

A260/A280

A260/A230

Left Fragment

78.0

1.86

1.85


Discussion

3/7/2024

The math portion of assembly was a bit difficult because we initially did not know how to treat the four fragments. We were unsure if we should treat the vector as one portion and the other three fragments as another portion in order to get our total volume. The other option we had was to treat each fragment as an individual portion that will add up to the total volume needed. In the end, we settled on the above calculations which would treat the vector as one and the three fragments as another. 

The nanodrop readings were lower than our original readings by quite a bit. We are unsure as to what caused this difference. We tested our left fragment but unfortunately, we did not have enough volume of the other three fragments to check them on the nanodrop as well. After talking, we decided that we will need to go back and redo PCR on all four of our fragments. The original nanodrop readings of our clean PCR product from the Left Fragment are: 


Sample 

ng/µL

A260/A280

A260/A230

Left Fragment 

82.4

1.86

2.28


Conclusion

We had more setbacks than we expected. Unfortunately, we were not able to do Gibson Assembly and we will have to wait until after spring break to do anything else. After spring break, we will be going back and doing new PCR reactions for all four of our fragments due to the inconsistent results that we were getting from our clean PCR products. 

Comments

Post a Comment

Popular posts from this blog

Spring 2024: Week 14 - April 29, 2024 - May 3, 2024

Image
  Introduction  Last week we attempted Gibson Assembly for the first time. As we interpreted the results closely, we seem to have been successful. We had a faint band at around 2,500 bp and our fragment was 2,527 bp. Since the band was faint, we are going to attempt to amplify it this week. Methods 4/29/24 We kept our broth culture of D. metalli in the fridge and the biofilm has continued to form. Since it is one of the few species in the Deinococcus genus that forms a biofilm, we were interested in seeing it under the microscope. We decided to do a gram stain on it.  4/30/24 In order to amplify our Gibson product, we decided to run it through PCR again. This time, we did Q5 PCR to get a higher yield than what we previously got. We ran two different samples. The first sample was 48 µL of mastermix + 2 µL of our 3-way Gibson that had been put through PCR last week. The second tube was the original Gibson product that had not been put through PCR.  At the sam...
Read more

Spring 2024 Week 2: January 29, 2024 - February 2, 2024

Image
  Introduction:  This week we continued our work with E. coli pRHAM. Last friday, we completed a plasmid extraction in order to get the pRHAM plasmid out. This week, we focused on confirming that we successfully extracted the plasmid before moving onto using that sample in PCR. If both of these go smoothly, we will be able to begin assembly, using Gibson Assembly, at the beginning of next week.  Methods: 1/29/2024  Using gel electrophoresis was our way to confirm if we had extracted the pRHAM plasmid. We mixed 2 μL of UV blue dye with 10 μL of our plasmid sample in an Eppendorf tube. We spun our tubes down in the pop spinner to make sure it was all one homogenous liquid. Since the plasmid’s size is 947 kb, we added a 1 kb ladder into one of the wells so that we could compare the size of our band to the ladder. We let the gel run and checked it under the UV light.   1/30/2024 Using our plasmid sample, we ran a short-amp PCR. We mixed 8 μL of mastermix ...
Read more

Spring 2024: Week 15 - May 6, 2024 - May 10, 2024

Image
  Introduction  During our last week in the lab, we attempted to perform Gibson Assembly with the 3-way fragment we previously assembled and the vector (pRAD1 plasmid). We are also preparing for the summer semester when we will also continue our work with D. metalli .  Methods  5/6/24  We are running out of pRAD1 plasmid extractions. We will need to extract more since we will continue to use it for this project and also for our D. metalli project. We inoculated E. coli pRAD1 into a flask of 25 ml of LB broth. We added ampicillin into the broth before inoculating. Since we plan on extracting within 24 hrs, we placed the flask in the shaking incubator overnight.  We also performed Gibson Assembly. This time we are attempting to assemble the 3-way fragment and the pRAD1 vector. If it assembles correctly, we will have a fragment that is 3,500 kb. We put the 3-way fragment and the vector in a PCR tube with the Gibson mastermix. The tube was incubated at 50℃ f...
Read more