Week 6-Semester 2. Reference Guide #1

Hello! This week again finds that my DNA extractions and subsequent absorbance readings conducted using the spectrophotometer will fall on Friday, the day after this blog is due. As a result, any data collection and methodology conducted this week will appear in a later blog post.

Part of my research project this semester is to become personally proficient in the use of all lab equipment and also guide any students that follow in the operation of the previously mentioned (on this blog) “lost” spectrophotometer by compiling detailed instructions for usage. Therefore, this week’s post will be a brief process analysis about using the Helios spectrophotometer to take live UV absorbance ratio readings.

The reader should be cautioned that there are additional methods for taking readings, such as programming and saving fixed methods, and other nuances within the settings menu that may affect sequence progression of individual wells and absorbance ratio levels. This additional information will be included in future blog posts. In the interim, I have pre-set and saved the settings needed to conduct the following protocol. If implemented as described, without any changes to the settings menu, the user can quickly and easily take accurate live absorbance readings.

Quick guide to live reads:

Notes/overview:

·         The Helios needs 10-15 minutes to warm up before being used.

·         The user should not leave the lid open or peer into the bay for an extended length of time; safety goggles with UV protection are recommended.

·         Bacterial DNA solutions should be at a 1µl DNA/99µl diluting liquid ratio (total volume 100µl). Molecular biology-grade water works best due to its purity.

·         The control sample should be 100 µl of the same liquid used to dilute the DNA sample.

·         Due to an inherent flaw in the design of the Helios, both control and DNA samples may need to be increased to 200µl total volume each to improve data collection.

·         One reading will be taken of the control sample @ 260 nm to set a baseline.

·         Each DNA sample will be subjected to three nm readings in the following order: 260 (to measure DNA presence), 280 (to measure protein contamination), and 320 (to measure turbidity, i.e. additional contaminants). It is important to begin with the 260 nm setting, as any DNA will be degraded by the higher wavelengths, and accurate measurements will be negatively affected by using a different progression sequence.

·         The user should record absorbance ratios given by each nm level in order to later calculate DNA purity and concentration.

Begin:

1)      Insert control cuvette into well #1. Place cuvettes containing DNA samples in wells 2-7 as needed.

2)      Make sure well #1 is aligned in anterior position (between UV bulbs).

3)      Program control sample:

a)      Set UV nm @ 260 nm for zero-base reading by pressing “nm” on LED display.

b)      Type desired nm wavelength (260) in display. Hit enter.

c)      Press zero-base key to set read at zero.

d)     LED display should read 0.000 A 260 nm. Record data by writing it down (or print by pressing print key on LED display.)

4)      Program DNA samples:

a)      Use right arrow key to advance to desired well.

b)      Set initial read @ 260 nm as outlined in step 3.

c)      Display will show absorbance reading @ 260 nm. Record data.

d)     Re-set nm @ 280 nm. Record data.

e)      Re-set nm @ 320. Record data.

f)       Repeat steps 4a-e for additional wells.

The procedure is as simple as that! Good luck with your samples!

Please enjoy this excellent website from the Smithsonian about DNA. Website is also photo credit:

 http://www.genome.gov/smithsonian/