Spring 2024: Week 5 - February 20, 2024 - February 23, 2024

 Introduction: 

In previous weeks, we have not been able to get good results when cleaning our D. rad PCR product using the Monarch RNA/DNA Cleanup Kit. We have been able to clean up our other three fragments using the kit but we were not able to get the desired results when cleaning up our left fragment. Jonathan suggested that we should look into an alternative method of cleaning up our sample: Alcohol Precipitation. 

Methods: 

2/22/2024 

In order to be able to do alcohol precipitation, we had to run another PCR reaction using our D. rad genomic extraction sample to get our left fragment. We wanted to yield a higher volume of our sample compared to previous PCR reactions that we have run because we will lose volume when doing Alcohol Precipitation. 

We mixed 36 μL of mastermix with 4 μL of our sample. We used two different genomic extraction samples labeled “MC” and MC2”. These samples had our highest ng/μL, A260/A280, and A260/A230 values. The ThermoCycler ran at the following times and temperatures: 

  1. 95℃ for 4 minutes 

  2. 95℃ for 30 seconds 

  3. 60℃ for 30 seconds 

  4. 72℃ for 30 seconds 

  5. Repeat steps II-IV 30 times 

  6. 72℃ for 7 minutes 

  7. 72℃ for ∞ (this is a hold) 

Results: 

D.rad Left Fragment PCR 

Sample

ng/μL

A260/A280

A260/A230

MC

277.1

1.87

1.79

MC2

261.3

1.88

1.74

Nanodrop results from D.rad left fragment PCR with a higher volume yield. 

Conclusion: 

2/22/2024 

The PCR that we ran to get our left fragment went successfully. We were able to get a higher volume that we will need for Alcohol Precipitation next week. The plan to do the Alcohol Precipitation protocol was pushed back until next week due to scheduling conflicts.

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