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

 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 with 2 μL of Kalen’s plasmid sample and another 2 μL of his second plasmid sample. This gave us a total volume of 22 μL. The ThermoCycler times and temperatures were as follows:

  1. 95°C for 4 minutes 

  2. 95°C for 30 seconds 

  3. 58°C for 30 seconds 

  4. 72°C for 30 seconds 

  5. Repeat steps II-IV 30x

  6. 72°C for 7 minutes 

  7. 10°C hold 

2/1/2023

In order for us to begin Gibson Assembly, we needed to clean up our PCR product. We followed the following steps.

  1. Dilute sample with DNA Cleanup Binding Buffer according to the table. Mix well by pipetting up and down or flicking the tube. Do not vortex. 

    1. A sample volume of 20-100 μL is recommended. For smaller samples, adjust the volume with TE.


Sample Type 

Ratio of Binding Buffer : Sample

Example 

dsDNA > 2kb 

(plasmids,gDNA)

2 : 1

200 μL : 100 μL

dsDNA < 2kb

(some amplicons, fragments)

5 : 1

500 : 100 μL

ssDNA > 200 nt

7 : 1

700 μL : 100 μL

Highlighted is the ratio that we used. 

  1. Insert column into collection tube and load sample onto column. Spin for 1 minute then discard flow through.

  2. Re-insert column into collection tube. Add 200 μL DNA Wash Buffer and spin for 1 minute.

    1. Discarding flow through is optional

  3. Repeat step 3 

  4. Transfer column into clean 1.5 mL microfuge tube

    1. Make sure the tip of the column does not come into contact with flow through. If you are unsure, re-spin for 1 minute. 

  5. Add ≥ 6 μL of DNA Elution Buffer to the center of the matrix. Wait for 1 minute then spin for 1 minute to elute the DNA.

Results:

1/29/2024

 

pRHAM plasmid on a gel. From left to right: 1kb ladder, KM1, KM2, MC1, MC2

1/30/2024 



ng/μL

A260/A280

A260/A230

KM1

367.7

1.84

1.90

KM2

493.3

1.85

2.04

PCR product nanodrop results 

2/1/2024 



ng/μL

A260/A280

A260/A230

Left Flank 

2.6

2.23

1.15

Right Flank

24.7

1.93

1.85

pRAD 1

38.3

1.96

2.15

pRHAM

1.6

104.67

2.07

PCR Product cleanup nanodrop results 

Conclusion:

Our right flank and our pRAD 1 PCR product had good results and we will be using them when it comes time for assembly. Our left flank and our pRHAM PCR product did not have good results. The values were too low and we will have to clean up our duplicate samples that we have of them. If those have better results, we will be using them to assemble. If they do not have the values that we are looking for, we will need to take a few steps back and go back to the extraction process. 


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