I'm having trouble obtaining clear PCR bands for DNA fragments ranging from 907 bp to 655 bp. I've tried various methods, including:
Please provide suggestions on how I can obtain clear PCR bands for my products.
Make sure the agarose gel is completely set and cooled completely. Use the thinnest comb that you can find and run the gel at half the voltage that you are now using. Can you attach a picture of the gel result that you are getting which will help identify problems. How much (ng) dna is in each pcr reaction
First, you need to slow down and stop changing so many variables. Pick ONE sample of plant tissue & the actin primers. Borrow a sample of DNA from a colleague that you know amplifies well using the actin primers. Include a negative control with just water.
Let us know what you see.
Paul Rutland Sir, kindly find the attached gel image and help me to correct the issue. Thank you
It is still useful to know how much dna you are adding to each reaction and to also use a sample of dna from another researcher. Also the OD260/280 will help. One sample is trying to work which suggests that the primers are ok bu the problem may still be with either the quality or quantity of the dna being amplified
Thank you Sir Paul Rutland for replying. Your point is duly noted. The nanodrop reading of the rice DNA sample is 2116 ng/ul, and A (260/280) is 2.16. The CTAB method was used to isolate the plant DNA. I had taken a 1 ul of diluted DNA sample (200 ng/ul) for PCR. The Absorbance >1.8 for DNA indicates RNA contamination is there but I do not think just because of RNA contamination DNA amplicon will not be polymerized. Please correct me if I am wrong. regards
I agree entirely Viraj that there is rna present which make accurate adding od dna more difficult but even allowing for this I think that 200ng of dna is too much and that you run the risk of the sample having pcr inhibitors present in the added dna. I would take the sample that looks like it is trying to work and dilute it 1;2 and 1;4 and 1;8 and do the same with one of the failing samples. The logic is that diluting the dna also dilutes the pcr inhibitors and the pcr works more efficiently so less dna but also less inhibitor means more amplified dna
What about the ratio of your DNA?
What do you think about the PCR condition?
If these are correct then you will get sharp bands.
You may also use fresh buffer for gel run.
Abdus Sabur also you can use a fresh prepared TAE buffer for gel preparation
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