I started actually working in the lab and performed DNA Extractions from blood samples of a family with type 1 diabetic mother, non diabetic father, non diabetic daughter and a pre-diabetic kid.
Protocol:
- Pour all the blood from one PAXgene blood DNA tube into a processing tube containing Buffer 1 (lysis buffer: breaks opens all the cells, and separates DNA, RNA, and Protein)
-Centrifuge for 5 min.
-Discard the supernatant and add Buffer BG2(Wash buffer, washes away all the cells that were not seperated)
-Centrifuge for 3 min. and discard the supernatant
-Add Buffer BG3 (Digestion Buffer) breaks down all the proteins and RNA in the sample.
- Place the tubes into a water bath for 10 minutes. (The sample changes from light red to light green, which is how we tell that the protein and RNA has been digested.)
-Add isopropanol and invert the tubes 20 times, after which we can see white DNA strands clumping up together, and is really visible.
-Centrifuge for 3 min and discard the supernatant. Leave the tube inverted on an absorbent paper
-Add 70% ethanol and centrifuge again. Discard the supernatant
- Leave the tubes open on an absorbent paper to remove ethanol. This process allows the ethanol to dry out from the pellet and eliminates excess ethanol.
-Add 1 ml of buffer BG4(Resuspension buffer: Adds all the necessary molecules back into the sample with just DNA alone) and incubate in a water bath for 1 hour after which incubate under room temperature overnight.
- Collect data the following day using a Nano-Vue spectrophotometer.
My experiments so far have been successful, because all the data I have collected falls under the designated ranges.
Protocol:
- Pour all the blood from one PAXgene blood DNA tube into a processing tube containing Buffer 1 (lysis buffer: breaks opens all the cells, and separates DNA, RNA, and Protein)
-Centrifuge for 5 min.
-Discard the supernatant and add Buffer BG2(Wash buffer, washes away all the cells that were not seperated)
-Centrifuge for 3 min. and discard the supernatant
-Add Buffer BG3 (Digestion Buffer) breaks down all the proteins and RNA in the sample.
- Place the tubes into a water bath for 10 minutes. (The sample changes from light red to light green, which is how we tell that the protein and RNA has been digested.)
-Add isopropanol and invert the tubes 20 times, after which we can see white DNA strands clumping up together, and is really visible.
-Centrifuge for 3 min and discard the supernatant. Leave the tube inverted on an absorbent paper
-Add 70% ethanol and centrifuge again. Discard the supernatant
- Leave the tubes open on an absorbent paper to remove ethanol. This process allows the ethanol to dry out from the pellet and eliminates excess ethanol.
-Add 1 ml of buffer BG4(Resuspension buffer: Adds all the necessary molecules back into the sample with just DNA alone) and incubate in a water bath for 1 hour after which incubate under room temperature overnight.
- Collect data the following day using a Nano-Vue spectrophotometer.
My experiments so far have been successful, because all the data I have collected falls under the designated ranges.
Nikki what are the designated ranges for the experiment?
ReplyDeleteThe A260/A280 (wavelength from nano vue spectrometer) should be between 1.7-1.9 and so far all my values are around 1.8. Also, the DNA concentration should be between 150-500 micro-grams and all my values have been between 220-340.
DeleteHave you acquired any results so far with the DNA extractions you've been working with?
ReplyDeleteYes, I've performed a total of 4 extractions so far for which my values have been between 220-340 micro-grams of DNA and the ideal range is between 150-500 micro-grams. After I complete all the extractions, I will perform gel electrophoresis and site specific methylation using them to target and identify specific genes.
DeleteNice yield! You seem to be enjoying your work and learning new stuff. Take pics of the DNA gels you run and other lab set up while doing your extractions. You could use these for your presentation.
ReplyDeleteYes, I love this lab. Everyone is great at teaching me everything, and they all know what they are doing. I performed my first gel on friday!! It was a great experience.
Delete