Lab 4: DNA Extraction (2/6/19)
Objective:
The objective of this lab is to extract DNA from the samples we have collected from previous labs. We also have to study our leaf sample and write down observations about it.
Purpose:
The purpose of this lab is to extract DNA from our Soil samples and our culture ciliate samples. We will also try to correctly identify the species of our tree that we collected our soil from.
Materials:
- Leaf Sample
- Centrifuge
- Tree Dirt Sample
- Ciliate Culture
- Chelex
- Micro centrifuge tube
- Micropipettes
- Micropipette tips
- Hot water bath
- Cold water bath
- Vortex
Procedure:
Chelex Extraction Procedure:
- Transfer 1mL of ciliate culture to a microcentrifuge tube
- Label your tube with soil identifier, and which ciliate culture your extracting from
- Centrifuge the sample at 6000g. discard the supernatant. Do this for 5 minutes
- Add 200 microliters of 5% Chelex 100 to pellet. Vortex for 1 minute
- Use large micropipette tips
- Incubate for 30 minutes in 56 degree water bath
- Boil for 8 minutes in a hot water bath
- Vortex it again for a minute
- Centrifuge at 16000g for 3 minutes
- Transfer supernatant to a clean microcentrifuge tube, don’t transfer any chelex beads
- Label the tube with soil identifier, date, well culture, and Chelex
Results/Data:
Soil Metadata:
- Sand: 2.3 cm (57.5 %)
- Silt: 1.0 cm ( 25% )
- Clay: 0.7 cm ( 17.5 % )
Total: 4 cm
Non-Flooded plate:
- Before: 15.4 g
- After: 14.9 g
- % Water Content: 5.6 %
DNA Extraction:
- Used about 400 mL of Culture
- Contained Ciliates in the Soil and in the culture
- Bacteria were present
Leaf Observations:
- Had a giraffe like patte
- Dark Green color
- Light green color on the lines
Conclusion:
After this lab, we were able to complete the DNA extraction of the culture using Chelex, so we will continue this for next weeks lab. The culture was difficult to observe, but ciliates were finally observed above the 10x magnification. I forgot to vortex the sample after adding the Chelex so I vortex the sample after adding the Proteinase K.
Future Steps:
I will continue to follow the procedure to extract DNA from my culture for next weeks lab. Once we are able to extract DNA from our ciliate culture, we can continue to follow the procedure of correctly identifying what kind of ciliates we have using DNA metabarcoding.