Hey guys,
I have some technical question to ask you.
I am using a Zymobiomics Miniprep DNA isolation kit for my samples. I eluted the DNA in 20ul DNAse/RNAse free water.
Then I performed PCR with 16S V4 primers and 5ul of eluted DNA. The gel of the PCR product is attached.
1st three lanes of the gel are from the PCR product of the DNA obtained from Zymo kit and then other 4 lanes are native DNA from Zymo, next 2 are positive controls and last is 1kb ladder.
Now I have 2 questions
1. Why we do not see any signal of native DNA obtained by Zymo kit (Probably we do not have enough cells in our sample which is giving us very low amount of DNA to visualize in the gel, or something else?)
2. After PCR amplification with 16S V4 primer we expect to get some DNA band of 250-350bp but we get much higher band size (1st three lane of the gel picture attached).
What could be the probable reasons and can we proceed to MiSeq library preparation with such kind of PCR product?
what are your positive controls? I can see smears on your gel image. Are you sure that your elution water is free from nucleases? Did you try to quantify your DNA using a Nanodrop prior to running the agarose gel?
I think it is a good idea to submit your PCR products for sequencing.
I do not know if you will further purify the products in follow the steps, if you further purify the product using beads, I think it is OK. Because this phenomenon occur in my previous experiment, it did not affect my DNA library quality.
Hi, Prashant Singh ,
1. Smearing is seen in your pcr product, so recommend to purify the genomic DNA first, when DNA contain lager amount of protein or with RNA's may cause such smearing.
2. Tm temperature for primers (You have for pcr) is not accurate, so recommendation for a gradient pcr with the primers. By gradient pcr you will find the accurate annealing and greater yield in pcr.
Hope you can find good result and analysis. Good luck!
Best regards,
nahid Islam
@Alemu Yes I did nano drop to quantify. My positive controls were the DNA from last experiments which showed a required fragment size. Yes, elution is done with DNAse/RNAse free water (Provied by ZymoKit). Thanks for your input
@Shixing Thanks alot for the suggestion.
@Nahid Thanks for your input as well. I am not purifying the microbial DNA only from the environmental samples prepared by washing leaf. Anyways, I will try to modify the Tm and will see the results.
Thanks all
you are using one quarter of your dna in a pcr reaction...that is probably too much. Try measuring the amount and using much less. You have a bad primer dimer band at the bottom of the gel. Try using a hot start enzyme and less primer. You should run a negative control with no dna to detect post pcr contamination which often produces high size product.
You do not expect to see much sample dna in a gel as it is usually degraded and smeared over a wide range. You should try running a temperature gradient with your primers as Sikder says which should remove much of the unwanted high size product. I would not prepare a library with most of your product not looking like the correct product
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