Spring 2024: Week 4- February 12, 2024 - February 16, 2024

Introduction: 

The main goal for this week was to finish acquiring all of the fragments and pieces that we need in order to move on to Gibson Assembly. We need clean PCR products of both of our left fragment and our pRHAM.

Methods: 

2/12/2024

We ran two different gels so that we could confirm that we had extracted the pRHAM plasmid and also successfully got the left fragment from our D.rad PCR. To do this, we made two separate 1% agarose gels and set them up in two separate apparatuses. For the pRHAM gel, we pipetted our 1,000 bp ladder into the first well. Then, we mixed 10 μL of our plasmid sample with 2 μL of UV dye. Since we had four total reactions when we did the plasmid extraction, we loaded four wells of our sample. This gave us a total of five wells. 

For our D.rad PCR product, we loaded our 100 bp ladder into the first well. We had four total reactions from when we did the genomic extraction so loaded four more wells. Each well contained 10 μL of our D.rad PCR sample sample and 10 μL of UV dye. We had a total of five wells. 

2/13/2024

We attempted trial #3 of our product cleanup. We are attempting to clean up our D.rad product from 2/8/2024. After talking with Dr. Tuohy, we decided to follow the original protocol and use 6 μL of Elution Buffer instead of the 10 μL that we attempted the last time. We checked the sample on the  nanodrop after doing the protocol and then added another 6 μL of Elution Buffer (into a new collection tube). We checked that sample on the  nanodrop and compared it to the first sample which only contains 6 μL total of Elution Buffer. We did this two more times, checking the samples on the nanodrop in between. He also asked us to save our flow through that the original protocol asks us to discard. We checked those on the nanodrop as well to see how the values compared to the clean product. 

We also ran PCR with our pRHAM plasmid that we previously extracted from E.coli. Since our gel confirmed that we extracted the plasmid, we were able to move onto PCR. We ran the ThermoCycler at the following temperatures and times: 

  1. 95℃ for 4 mins  

  2. 95℃ for 30 seconds 

  3. 58℃ for 30 seconds 

  4. 72℃ for 30 seconds  

  5. Repeat steps II-IV 30x 

  6. 72℃ for 7 mins  

  7. 10℃ for ∞ (this is a hold)

2/14/2024

We did our fourth PCR cleanup. This time, we worked with our pRHAM PCR product. We decided to follow the plan that we came up with with Dr. Tuohy except this time, we did not save any of the flow through. We added Elution Buffer until we had good A260/A280 and A260/A230 values. 

We also ran a gel to see our pRHAM PCR product and confirm that we have gotten what we are looking for. We added 10 μL of our sample and 2 μL of UV dye. 

2/15/2024

We attempted another PCR product cleanup on our left fragment. The left fragment comes from D.rad and we have been having trouble getting good values of it after cleanup. This time, we decided to use the tube labeled MPB. As usual, we mixed 6 μL of our PCR product with 14 μL of TE Buffer. When it came time to add the Elution Buffer, we followed the protocol and added 6 μL. We took a nanodrop reading of the clean sample and then added 6 more microliters of Elution Buffer. 

Results:

2/12/2024 

2/13/2024

Left Fragment Nanodrop dsDNA 

Sample 

ng/μL

A260/A280

A260/A230

MC 

41.7

1.81

1.67

Waste #1

9.4

1.47

0.20

Waste #2

3.3

1.26

0.21

Waste #3

2.9

1.45

0.17

12 μL Elution Buffer

7.4

1.77

1.75

18 μL Elution Buffer

0.4

0.94

0.45

24 μL Elution Buffer

0.9

1.00

0.51


2/14/2024


pRHAM PCR Product Cleanup (AN1)

Sample 

ng/μL

A260/A280

A260/A230

AN 1 (Reg. Protocol)

67.2

1.91

2.24

AN 1 (12μL E.B.)

6.7

2.37

2.71


pRHAM PCR Product on a Gel 

2/15/2024

I made a mistake when it came to adding the other 6 μL of Elution Buffer. I forgot to move the column into a new collection tube/eppendorf tube and added the extra Elution Buffer into the same sample. The results may not be as accurate/reliable as they could have been.


Left Fragment PCR Product Cleanup

Sample 

ng/μL

A260/A280

A260/A230

MPB (Reg. Protocol)

39.7

1.68

1.11

MPB (12 μL E.B.)

28.7

1.61

0.97


Conclusion: 

This week we had some trouble getting the results that we wanted but we were able to experiment to see what worked the best for us. When we ran our pRHAM plasmid and our D.rad PCR product on a gel, we got bands of confirmation.We were able to get good results for our pRHAM PCR Product when we cleaned it. As for the left fragment, we might be looking at trying alcohol precipitation next week as a way of cleaning it. 

 

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