I have isolated DNA from 382 plants without running them on gel . I thought i will run them together after one month of completed extraction i found a long band of smear on my gel for all of my samle . I want to run SSR marker for these sample what i dont know is this possible or not . Nanodrop conc. of all of my sample is ranging from 500- 2000 ng/microlitre with A260/A280 ratio of 1.8 -2.02. lease tell me what should i do ,i cant perfom dna ext6raction of this much sample again . My gel images are attached below
1. How did you store your DNA samples? In what solution?
2. I would suggest you run your samples again (just use 5-6 same samples to try), using a freshly prepared TAE buffer. Make sure your wells are not broken. Reduce your running power. And, include a DNA markers on the side. After that, please update us.
i store my sample in -20 degree dissolved in 100 microlitre TE buffer ph 8. I used 0.5 X TAE buffer to run gel electrophoresis. I
extracted DNA using CTAB method . The protocol that i have followed is is 65 degree incubation of 1 hour after grinding through silica beads ,then Rnase treatment for 1 hour in 37 degree , then 2 times washing with Chloroform :Isoamyl alcohol, followed by adding 5M ammonium acetate-ethanol for precipitation overnight, washing with 70% ethanol and air dying in 37 degree incubator with open caps for 2- 3 hour .
ther is rna present and also degradation of the dna but so long as many of the degraded dna fragments are longer than your expected pcr amplimers I would expect pcr to work well on these samples. People are amplifying ancient ( m0re than 6000year old) and cell free circulating dna both of which are broken up into small fragments and are successful
Should i mix RNAse A in my DNA samples ? if yes then how much concentration from 10 mg/ml stock ? Do i need to remove that RNase before PCR amplification ?
use about 50 µg / ml RNASE A for 0.5ml of crude DNA preparation) and digest 37c for 30mins. It will not have any effect on pcr but although the rna is broken up the bases will still absorb in the UV range so if you need to measure the amount of dna then a purification step is a good idea
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@ Shubham Bhardwaj,
Why did you use 0.5x TAE buffer to run your gel, instead of 1x TAE buffer??
Could you run it (use 4-5 samples) one more time as I suggested above with 1xTAE, and then update us? Also, remember to make your gel with the same buffer (1xTAE).
@Yuan-Yeu Yau After running on 1x TBE buffer, my samples still showing degraded DNA. I have also given them RNAase treatment but there is no big difference.
i think problem is in my crushing machine , when i extract dna from six fresh plant samples through both liquid Nitrogen and through silica beads crushing machines with same protocol and same solutions . Results are contrasting to each other, i got clear bands without smear with liq. N2, but degraded bands with crushing machine. So in this way i reached to conclusion that due to some mechanical shearing my DNA got degraded even if crushing happened in ctab extraction buffer .
What was the speed and time of crushing DNA machines? Usually at 30 rpm speed and 2 minute the sample degrade. So reduce the speed whenever you are using crushing machine in range of 20-22 and for 1 minute crush it at one side and now invert the lid and again crush for 1 minute at other side.
If your sample is degraded then dilute the sample as we are using for PCR and again run the sample in 0.8% gel. Or check the 1-5 smear DNA for PCR.
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