Common lab tools and their coinciding procedures

 Introduction:

This past week, the lab analyzed each project and made decisions on what direction to move forward. I was unable to perform any lab work due to not being on any project at the time. While awaiting for an open research opportunity, I decided to try to gain a fuller understanding on how to use some of the common tools found around the lab. I learned the procedure to take proper nanodrop readings. 

Methods:

1. With a Kim tech wipe and deionized water, clean the parts of the nano drop that will touch the subject along with the pedestals.

2. Soecify OD values.

3. Pipette 1-2µL of blanking solution onto lower pedestal and lower the arm.

4. Tap the blank button and wait for a measurement to be complete.

5. Lift arm and clean both pedestals with a new laboratory wipe.

6. Pipette 1 to 2 µL of simple solution onto the pedestal and lower arm.

7. Start sample measurement by tapping “measure”.

8. When sample measurement is complete, the spectrum and reported values will be displayed.

9. When finished measuring samples, tap “end experiment.”

10. Lift arm and clean both pedestals.

11. Adjust dilution of sample until you get OD value of 1.

Results: 

Results from nanodrop readings will display as a graph, showing a relationship between 10mm absorbance rate of UV spectrum, and the amount of dsDNA. Two purity ratios will be provided. One displays the ratio of corrected absorbance at 260nm to the corrected 280nm. The other ratio displays corrected absorbance at 260nm to the corrected 230nm. 

Discussion: 

The nucleic acid concentration is calculated off of beers law equation, using corrected, 

nucleic acid absorbance values. The nanodrop is a valuable tool for microbiology lab work, since the 1-2µL of sample required makes it ideal for a small sample size. The A260/A280 ratio indicates protein contamination. For DNA readings, we want to get a reading of a ratio as close to 1.8 possible, since that is what’s found with pure DNA. Pure RNA would give a reading of 2.0. A260/A230 ratio indicatates contamination of organic compounds, salts, or phenol. Purity ratio of 1.8-2.2 is generally accepted as “pure” for both DNA and RNA. Samples must be homogeneous when pipetting onto pedestal, since viscous or particulate samples can impact results. This data allows for further applications, such as sequencing, PCR and qPCR. Next week in lab, I plan on joining a group doing transformations, and learning procedures surround plasmid extraction and transformation. 

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