Spring 2024: Week 6 - February 26, 2024 - March 1, 2024

 Introduction 

In previous weeks, we have not been getting successful results using the DNA Cleanup Kit for our left fragment. We have used the same kit to clean up the other three fragments. We decided to try an alternative way of cleaning up our left fragment by doing alcohol precipitation.   

Methods

2/26/2024 

We did alcohol precipitation to try to clean up the left fragment. We followed the following protocol:

  1. Add the following to your sample:

    1.  0.1 vols of 3 M sodium acetate 

    2. 2.5-3.0 vols of ice cold 100% ethanol

    3. Vortex to mix thoroughly

  2. Precipitate at -20℃ overnight or -80℃ for 1 hour

  3. Centrifuge at full speed for 30 minutes 

  4. Wash pellet twice with 0.5 mL of ice cold 75% ethanol, spinning at 4℃ for 10 minutes each time

  5. Take ethanol out, spin quickly (10s top speed) to remove the trace amount of ethanol

  6. Air dry the pellet and resuspend in an appropriate volume of nuclease free water

*precipitating small amounts of RNA*

Glycogen 20 ng per sample may be added to RNA before precipitation to aid visualization when precipitating small amounts of RNA 

  1. Add 1 µL of a 20 mg/mL solution of Glycogen 

2/27/2024

Due to lack of time on 2/26/2024, we checked our PCR product the next day. We put it in the -20℃ to store and when we pulled it out, we put it on ice. We also inoculated our E. coli pRHAM and our D. rad to keep our cultures growing. If for any reason we need to go back and extract from them, we have them ready to go. 

2/28/2024   

I checked the previously inoculated bacteria (2/27/2024) on the nanodrop to see the growth. We also spent time trying to work out the calculations needed for Gibson Assembly and becoming familiar with the protocol. 

Results 

2/27/2024

We checked our left fragment on the nanodrop to see if the alcohol precipitation was able to clean it successfully. 


Sample 

ng/µL

A260/A280

A260/A230

MC

63.0

1.85

1.70

MC2

82.4

1.86

2.28

Left Fragment Clean PCR Product 

2/28/2024


Sample 

OD600

E. coli pRHAM 

Ino. 2/27/24

3.62

E. coli pRHAM Nanodrop Results 


Sample 

OD600

D. rad 

Ino. 2/27/24

2.87

D. rad Nanodrop Results 

Discussion

During the Alcohol Precipitation, we followed the calculations using the amount of sample that we had to begin with. We had previously done a PCR reaction that gave us 40 µL of product. We used 2 µL to check it on the nanodrop which left us with 38 µL. We did the calculations as follows: 

Sodium acetate: 0.1 x 38 µL = 3.8 µL

100% Ethanol: 2.5 x 38 µL = 95 µL

Since our pipettes only go up in increments of 0.5, we had to round up the volume of the sodium acetate to 4 µL.  We also added 1 µL of Glycogen to help us see the pellet. 

Conclusion

After doing Alcohol Precipitation, we were able to successfully clean up our left fragment. We are now able to use it for Gibson Assembly. We plan on assembling in the beginning of next week (3/4/2024-3/8/2024).

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