Sample: muscle tissue (~4mg)
Extraction Kit: Macheney-Nagel - DNA, RNA & Protein Purification Kit
DNA concentration (Biodrop) = ~20ng/µl
Gel: 1% Agarose, 100V, 40minutes
DNA ladder: 1kbp ladder
Volume of sample loaded= 5µl (~100ng)
Result: No visible DNA bands other than DNA ladder, no smearing and no DNA shown left in the well
Can this happened due to DNA degradation? If so, is there any extraction protocol to obtain high molecular weight genomic DNA?
attached gel:
lane 2: DNA ladder 1kbp
Lane 4,5,6 - samples
Dear Wai,
Can you share the A260/ A280 and A260 / A230 ratio?
Also, Make sure the blank solution used for conc. detection was the eluent used.
Dear Wai,
Have you checked the purity level of DNA at A260/ A280 ratio?
Sometimes, the DNA may get denatured while extraction procedure, giving a smeared appearance. Try to perform your experiment in cold conditions, or else used ice-cold solutions for your experiment. Also, do not repeatedly thaw your sample.
Also, if there are traces of proteins in your extracted sample, it may hinder the gel electrophoresis and give a smeared look.
First of all, thanks for the reply, Gargi Nanda and Joyyeta Mitra =)
More info about the extraction:
1. A260/280=1.8015
2. Blank used was the eluent (BE from the kit)
3. i didnt get any smear at all, it's all so clear...
(will attach an image later)
Thanks Arthur Burzynski. I will definitely going back to the protocols and revise according.
For gel electrophoresis, I've tried loading samples from other colleagues and there are clear DNA bands shown.
Is it possible to get good readings from Biodrop if there is no DNA from my samples? Can the reading only indicates presence of other proteins?
Try loading more volume of your sample. If there isn't any DNA, there shouldn't be any problem in trying to load max possible volume and be sure of its presence or absence. If at all there shows a band, you can quantify its concentration by measuring its intensity. Later you can know how much of tissue can yield what qty of DNA.
FYI: Minimum amount of DNA conc visible on electrophoresis is 50 ng.
Dear Wai,
I've seen some stain at the start of the sample row. More important, that if your sample is not homogeneous you need to load 300-500 ng of DNA to see a predicted smear.
Good luck!
Hello Wai,
1) As Artur pointed out 4mg is not a lot for this kit, my tests (used fin clips and tissue plugs) suggest that the optimum is typically ~25mg. I would try testing different amounts of tissue (if possible) and see if you can get more DNA.
2) As a couple of people have mentioned 20ng/ul can be hard to see on a gel and is on the lower-end of what the Biodrop can pick up. I truthfully would not trust a Biodrop reading for samples below 40ng/ul. Depending on who you talk to some trust as low as 5ng/ul, but if you are having issues visualizing the DNA and especially since this is not purified I would be more cautious with the readings.
One way to test this is to concentrate the sample and rerun the Biodrop and gel. I would suggest using Ampure beads since the will concentrate and remove contaminates from the sample, allowing you to have more accurate readings.
Also if you are using low concentrations like this I would use a Qubit to test the concentrations, easier to use, cheaper, and more reliable at low concentration, widely preferred for more NGS methods, see link below for a good discussion about NanoDrop vs. Qubit.
https://www.researchgate.net/post/Qubit_vs_Nanodrop2/1
Hope this helps.
Best Regards
Thank you all for the precious opinion on this question.
I've increased the starting material to 40mg of frozen muscle tissue, and allowed overnight digestion, using QIAGEN blood and tissue kit
I got some results from gel electrophoresis (DNA conc from Biodrop = ~40ng/ul, 2% agarose gel). Although the band did not appear very bright, but it's a good indication of genomic DNA with high molecular size and there is no smearing on the gel. =)
Will definitely try concentrating the samples and optimizing the extraction.
Could anyone tell me please what the expecting band size for gDNA would get from human tissue?
Hello,
Not sure if you got your problem sorted already. Had similar challenges before. One of the issues was on sample quality. I was working on blood samples collected in remote places on filter paper and seems like their quality got compromised somewhere along the way. whey I switched to fresh and more carefully handled samples the problem was sorted out.
secondly, I don't know how your kit works, but I use the traditional phenol chloroform method for whole genome sequencing on NGS and the DNA is most of the time great. What I ensure, out of bumpy experiences like yours, is to avoid any processes that can cause degradation. Slow/gently spinning/ shaking etc and cutting the tips of pipette-tips to make the mouth bigger and avoid shearing the DNA while pipetting are great tips.
cheers..
Thank you so much @Newton Otecko, I did change to another samples (frozen sample) and the result is good. =)
I heard that traditional method works well. I do have some blood and hair samples in hand, would you mind to share the protocol for DNA extraction from blood and hair? Thanks in advance.
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