I am currently working on dna extraction from soybean leaves. Months prior, I could get clean and clear bands of DNA extracts when viewed in the agarose gel. But this time around, when I checked the results, it showed intense bands at the end of the gel, which I assumed to be sheared DNA fragments. Any tips to prevent this? Thank you!
Hello Santos,
Having a look on your gel, I would like to suggest the following points :
1. If you are following a manual method of DNA extraction, please increase the duration of the lysis step. When the lysis is not perfect, the smaller fragments of nucleotides( fragmented DNA, RNA etc) pop up out of the cell very easily. The concentration of these nucleotides outnumbers to that of desired DNA, leading to an intense band at the bottom of the gel that you have shown.
2. After lysis step, be precautious if vortexing is required! It may shear your DNA if exposed for a longer duration of time.
3. Determine the DNA/RNA ratio by taking OD at 260 and 230 nm (for the sample you have shown in the gel)followed by RNase treatment to remove the unwanted bands.
Best wishes,
Bhuwan
Hey there
May be your DNA samples have got contaminated
Try to use DNA clean up kit to clean your DNA
You may try to change water which you use for the PCR cocktail - old/contaminated water often gives such picture on a gel. Hope that helps!
Hello Santos,
Having a look on your gel, I would like to suggest the following points :
1. If you are following a manual method of DNA extraction, please increase the duration of the lysis step. When the lysis is not perfect, the smaller fragments of nucleotides( fragmented DNA, RNA etc) pop up out of the cell very easily. The concentration of these nucleotides outnumbers to that of desired DNA, leading to an intense band at the bottom of the gel that you have shown.
2. After lysis step, be precautious if vortexing is required! It may shear your DNA if exposed for a longer duration of time.
3. Determine the DNA/RNA ratio by taking OD at 260 and 230 nm (for the sample you have shown in the gel)followed by RNase treatment to remove the unwanted bands.
Best wishes,
Bhuwan
Bhuwan Bhaskar thank you so much for the detailed suggestions. will try out those. again many thanks!
Hi Santos,
I do not think this is RNA contamination because You did not observe the shearing first time.
This is probably degraded DNA, which could be due to improper storage of DNA for long period of time (means inappropriate temperature) or due to some contamination.
Also make sure that your electrophoresis system is proper because it may be resulted due to very old and exhausted buffer and so on.
Please be gentle with your voltexing wherever it is recommended that you do so! looking at your gel, I AGREE WITH Faryal, for it doesn't appear to be due to RNA contamination. However, to be sure, use a protocol that incorporates RNase use for their digestion.
I think that 1- vortex 2- you should make crystallization very well in ice before gel step 3- make sure that all ingredients an tools clean
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