So I was testing genomic DNA extraction protocols for plant but I never get anything in my electrophoresis gels, I uploaded the pics, its the last three lanes.
The last protocol I used was from Chabi et al 2015 "A simple genomic DNA protocol plant systems" and it goes as follows:
- Take half of a young dry leaf and cut it into small pieces then grind it using a porcelain mortar and pestle in 400 μl of the extraction buffer (the mortar and pestle was previously washed with HCl 1N, left dry in a fume hood and kept at -20°C, this was in order to eliminate any previous DNA and the coldness was for easier grinding).
- The extraction buffer is made of SDS 1% and NaCl 0.5M only.
- Add more buffer until it reaches a final volume of 1200 μl in order to have enough homogenate to place into microfuge tube.
- Harvest the homogenate into 1.7 ml microfuge tubes.
- Spin (13,500 rpm, 4 min, RT) using a microcentrifuge.
- Transfer the supernatant into a new microfuge tube and add an equal volume of isopropanol (500 μl in our study) and mix gently by inversion.
- Place the mixture on ice for 5 min, I placed in the bottom part of the refrigerator, since its 4°C, I didnt have ice
- Spin (13,500 rpm, 4 min, RT) using a microcentrifuge. After this I got a greenish/brownish pellet, which I hope its from any remaining chlorophyl.
- Discard the supernatant and wash the DNA pellet with 500 μl 70% (v/v) ethanol.
- Spin (13,500 rpm, 2 min, RT) using a table microcentrifuge.
- Discard the ethanol. Blot away the excess ethanol from the pellet by inverting/placing it on a clean paper-towel.
- Let the pellet air-dry. Dissolve the DNA in 50 μl ddH2O and store it at 4 °C for immediate use. I used biomomecular grade water from Millipore, DNAse, RNAse free. Left it in 4°C to use it in my gel.
It's a simple protocol, but I got more DNA than in my previous tries, I wonder whats wrong since I've extractd genomic DNA from bacteria and it doesnt look like that at all, and Ive seen the genomic gels in Google and they look different. Anyone got any suggestion?
Thank you, :)
Hello,
maybe you have isolated intact genomic DNA, but its concentration is bellow ethidium bromide detection limit. What is the goal of your experiment? If PCR, I think you can proceed next, without need to do DNA electrophoresis. PCR requires minimal amount of genomic DNA (typically 10 ng is OK). Also you can measure DNA concentration of your isolated DNA on some sensitive device like Nano Drop or Nano Spectrophotometer.
By the way what plant are you working with? Some types of plants (e.g. conifers) may be hard to disrupt.
Best
Thats what my advisor tell me, and yeah its for PCR, but since I've worked previously with DNA I was skeptical. we dont have a Nano drop , :(, will recommend it to my advisor though. Thank you, I will still try PCR on those samples.
Currently Im working with Dolichandra unguis-cati.
You would not expect to see genomic DNA from plants on an agarose gel. If you don't have a nanodrop, you can use a spectrophotometer to check DNA concentration. But honestly, most folks just proceed right to the PCR without worrying about quantifying the DNA concentration.
In most cases plant genomic DNA can be easily visualize by agarose gel, but there are different protocols for extracting total genomic DNA! I think you got DNA, but it was in low quality!!
I advise you to use ready kits especialy those with silica filter columns that guarantee extracted DNA in adequate quantity and quality.
Best luck
Some of the bands and smears you are seeing on your gels may be RNA. Try doing an RNAse treatment of your samples and run equivalent amounts of treated and non-treated side by side.
If your pellet is still greenish/brownish you could try doing a second wash to clean up more of the residual chlorophyll and other substances. If your plants have lots of secondary metabolites in the leaves then the pellets can be a bit colorful and/or sticky, more washes will help.
What are you hoping to do with your DNA after the extraction?
I'm hoping to do PCR's, will see if an RNAse treatment eliminates the smear.
Thank you!
Just as an update, I did my PCR and I got product, so there was DNA after all, thank you all.
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