The picture of my result attached. The right two columns are negative and positive controls respectively. 260/280 of samples 1,4,5 are between 1.7-1.8 producing faint bands; while the 260/280 of the samples 3,4 are between 1.4-1.5 producing multiple bands. Why? Any advice?
Maybe use less primer as you obviously produce some unwanted PCr products and your primer clouds look very bright compared to positive control.. What is the difference between the samples ?
you might try to tighten up the PCR reaction by raising the temperature of melting..may be there are the non-specific amplicons that have nothing to do
Hi Norbert Bittner. Samples producing faint bands used self-made chemical solutions while samples producing multiple bands used chemical kit. And the 260/280 of them are about 1.75 and 1.5 respectively. Yes, the primer cloud is obvious.
You mean chemicals for extraction/PCR/whatever ? Please be a bit more precise ;)
if the samples with multiple bands have a lower 260/280 as compared to the ones with single bands then maybe the quality of your DNA is not good enough? if you have more sample/ DNA available maybe you could extract/ re-extract it using a commercial kit and see if it gives you a single band
The ratio 260:280 ideal for 100% of DNA is approximately 1.8. The lower this value greater contamination of your sample by proteins or salts. Consequently, worse the quality of your sample extracted. This may be one reason for the appearance of multiple bands. You could try to reduce the concentration of primer. This can reduce the excess primer on the base of the gel. A temperature gradient can help improve amplification. Excess DNA can also damage your PCR.
Hi, Norbert Bittner . I meaned the chemicals for DNA isolation. Thanks for others answering my question. I am keeping try.
Dear Callie,
The recommended DNA purity to start with any downstream analysis is 1.7-1.95 and some people mentioned you can use 1.6 purity DNA. For your multiple bands, I was about to tell you that the primers are not specific and change it but in this case which the purity is very low for both samples that might your samples are contaminated actually and not pure. Please check the reading of spectrophotometer at 230 and you may see it high I assume. For the faint band how much was the DNA concentration you used? You might need to increase the DNA. Try to measure your DNA with Qubit Flurometer if you have access to it because it will give you a consistent DNA concentration because it uses a fluorescent dye to bind to the dsDNA, while the UV can measure the DNA and nucleotides at 260 and 280nM.
Dear Amr,
Thanks for your answers.
For the multiple bands, A260/280 is about 1.6 and A 230 0.7.
For the faint bands, A260/280 is about 1.8, and A230 1.3.
The loading amount of DNA of each sample is 100 ng. However, the yield of DNA extraction is lower (around 1000 ng), and the concentration of DNA resuspending solution is 10-20 ng/ug. I think you are right. My DNA sample is not pure and its concentration is too low. Previously, I thought only the total loading amount would effect the results.
When I extracted DNA, very obvious DNA ropes could be seen after adding of isopropanol. However, after washed by 70% ethanol, I could not find them any more. I have repeated it 5 times. Could you please send me your protocol? Thanks
Hi Callie,
It depends really on your starting materials. Qiagen offers many kits depending on your application. For my blood samples I used QiaAmp Blood mini kit to extract the DNA.
I hope this will help.
All the best,
Amr
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