I have done PCR of 16S rRNA from bacteria genome. I used 27F and 1492R primer for the PCR. The concentration of the PCR product is small, so I want to repeat again the PCR with PCR product before as the template. Could it?
Of course, you can use PCR product as the template.
Please, read this conversation : https://www.researchgate.net/post/Is_it_necessary_to_perform_a_clean-up_step_on_PCR_products_for_nested_PCR_reactions/1
it may help you.
yes, you can do it, but using the same primers
best regards
Yes you can do it with same primers, used for amplifying the 16S rDNA with same reaction conditions. If your amplicon concentration is too high then dilute the PCR product 50 to 100 fold and use 5 microlitre tempalate (PCR product)/25 microliter reaction volume. or optimize accordingly.
It is highly recommended to purify and assay the PCR products. Often it works best with an internal primer rather than with one of the PCR primers.
Yes you can do it with same primers
Yes, you can use your PCR products as template in another PCR test using the same primers to increase the concentration of your PCR products
Hi everyone,
I have tried repeating a PCR using PCR products as template (using same primers) and it has worked well. I have even got good sequences from it. However I have never seen a publication mentioning this method so I want to make sure it's the right thing to do and it's publishable. If you know of such publication would you mind sharing it here? Many thanks.
This article might help you, and you can search for articles like this dealing with diminishing artifactual PCR products. I am trying to typing individuals for a highly polymorphic gene, and I have to be sure whether I find some new allele that it is not just an artifactual PCR product. So one of the recommended approaches is to do a second PCR with an aliquot of a first PCR.
https://www.ncbi.nlm.nih.gov/pubmed/20833233
What I usually do: when I use PCR products (1st reaction) as a template, I usually raise the annealing temperature of the primers (by a few degrees celsius - already increased by up to 6°C) in the second PCR, and this helps to make the reaction more specific and efficient.
HI there,
I'm coming into the conversation late, but was hoping someone could clarify something for me. In all of the instances mentioned above, did the initial pcr show faint bands or no bands? It has been recommended that i use pcr template in a new pcr for samples that showed no bands on the gel. I'm having a hard time conceptualising how this would work.
Hi Jessica,
Yes, you can do this but i'd recommend that your initial PCR product is very clean. Basically, you do the same as normal PCR with your set of primers but using your PCR product (as others have probably mentioned above). However, if your original PCR template is not giving any bands then there might be a problem early on with your parameters that you should probably address first. I have done this with PCR products that have weak bands and it has worked reasonably well.
Good luck
Typically, it is feasible. However, there is this one paper (which I cite below) that characterize the appearance of a ghost band with extremely high molecular weight, so much so that it would not leave the well, when purified PCR product was used as template DNA. The primary problem is that the abnormal ghost band appears instead of the expected product, which could not be found. However, this only happen if the annealing temperature is lower than the ideal temperature. Judging from the comments above, it doesn't usually happen, but it is good to keep an eye out.
Shenghe C, Wei S, Zhaoxi Z, Jingyang L, Minjie D and Haiyan S. A weird DNA band in PCR and its cause. J Plant Sci Mol Breed. 2016; 5:2. http://dx.doi.org/10.7243/2050-2389-5-2
Hey everyone, having some troubles as well, any recommendations would be appreciated.
Been trying to amplify trnfM and trnS1 from the chloroplast region of the plant we are researching. Have encountered some problems when attempting PCR.
Here's what's going on: I have run PCR on my samples multiple times, however, every time I only get 2-3 faint bands. I decided to use the faint bands (picture below, 9-24-19) as my template in the next PCR and received bands up around the wells (Picture below 9-30-19 chloro). Any thoughts for troubleshooting this?
You can use but gDNA is better as PCR by its nature may cause mutation
yes, I had tried it for 16SrRNA, and it went quite fine. The main issue here is that in an amplified PCR product, the concentrations of the desired DNA is quite high. So, before doing the re-amp, re amplification, you need to check the concentration of DNA in the product. I have to dilute my initial PCR product 10 times, in order to get a proper band. Although, wheather this method is scientifically accurate or not I don't know. but it worked for me quite well
Amlan Jyoti Ghosh how about ur DNA concentration in the product? I got 7.1ug/cm of my amplfied pcr product but the result(amplified pcr product as template) shows they are still leaving in sample groove.
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