I have problem when I want to PCR 16sr RNA gene of my unknown strain. I have extracted its DNA and did PCR by using lyophilized master mix tube for PCR. The result was good but I had an extra band in my gel electrophoresis. I did the PCR again but the problems exist. So I extracted the DNA by using another Kit and did PCR with another PCR pack. But the result was not good. I think my problem is in mastermix preparation or in my thermal program. Can anyone give me an advice to solve this problem?
This paper will help you understand the reasons better.
https://www.researchgate.net/publication/262560092_PCR_amplification_of_repetitive_DNA
Keep in mind most 16sRNA genes are multicopy and mostly located in concatemers, some of which might have additional seqment of DNA in between the copies. Even if your starin is pure, you might get such artefacts due to this multicopy arrangements. More explanations are in the paper.
So sequence both of your fragments and find out if you can explain it by such multicopy events.
Regards,
Bekir
Data PCR amplification of repetitive DNA
dear bakir
but the extra bad is smaler than 1500bp! so i want to ask can i send it to sequensing or not?
Anyone please correct me if I am wrong. You may have contamination. When I had this happen I had to check all my reagents were in good condition. Maybe try trouble shooting by changing your primers, dntps or mixes. If you have a hood I would also do my PCR prep in the hood as an extra measure. When you amplify 16S rRNA you run the risk of also amplifying any bacterial contamination that might be present. I would not recommend sending to sequencing until this is solved.
The size of 16s rDNA should not be significantly smaller than 1500bp if you are using primers that amplify the full length gene. Even if you have contamination from other bacterial DNAs, it should not give smaller bands. Terefore, your most likely problem is that the primers are miss-annealing somewhere else and producing these artefacts. Increasing your annealing temperature 3-5 degrees should reduce or eliminate this problem.
If increase in TM does not solve the problem and you are still curious to know what was the smaller band, you can indeed sequence that as well. However you should keep in mind that this PCR product might be generated by one of the primers alone instead of combinations. So, in such case sequencing with these primers will fail (actually will work but you will get overlapping signals from both directions and you could not get the correct sequence). Therefore I suggest first cloning the gel isolated version of that PCR product (TA cloning or TOPO blunt cloning) and sequencing it starting from universal primer binding sites located in the vector.
It might be also useful to post your representative gel image data so that you get a much better feedback.
Let us know the results please.
Bekir
dear all
i want to do gel recovery for 16sr RNA band and sent it to sequencing. can i get a good result?
Dear Muhtaq,
No need to worry, just do following to get your target results
1> First of all check the qauility of Taq ploymerase or use fresh stock of Taq by making manual PCR recipe, PCR thermocycler profile in attached link paper.
2> By morphological characterization, if your candidate strain belong to Pseudomonas or Enterobacter then follow all as in this paper for PCR.
https://www.researchgate.net/publication/260249394_Identification_and_Characterization_of_Rhizospheric_Microbial_Diversity_by_16S_rRNA_Gene_Sequencing
3> if you are in hurry and not able to get single band then excised the targeted band by Gel extraction kit (QIAGEN) and purified the extracted band by GeneJET PCR Purification Kit (Thermo Scientific). Then send it for sequencing, I experienced this and got good sequencing result.
Hope these things help you.
Stay happy
Article Identification and Characterization of Rhizospheric Microbia...
I generally do not have those problems. When a band is faint, I take 1 or 2 ul aliquot of the PCR product and reamplify using the same end primer and it produces strong amplification.
Although I don't know the exact cause of your extra band, I would suggest to run shorter cycle (let's say, 15 or 20 cycles) and use aliquot to reamplify in the next round of PCR.
Dear all
I did some of purposed changes in my master mix and thermal program and checked the primers and template DNA by non drop. One of my primers was contaminated and one of my samples has low concentration of DNA. I did the PCR with increasing annealing T 3 degrees and using more amount of DNA template. I got a good band but I had smear in gel electrophoresis. That belongs to protein impurities in template DNA.
How I can remove the smear?
Can I send these products to sequencing?
Thanks all
Hi Mishtaq,
Did you have a negative control in your amplification (i.e. a reaction with water as template)? If you did not, please, add a negative. By chance I have been discussing this with a PhD student, and we found out that the source of the contaminating band was the Taq itself! Taq is produced from bacteria, and depending on the purification procedure (usually lyophylisation. Ultra-pure Taq is purified by e.g. HIS-tag columns or FPLC) you can have bacterial contaminating DNA that is enough to prime amplification in your negative control.
We tested polymerases from three different suppliers, and one of them gave no band in the negative control (MyTaq from Bioline). If you find out that the Taq is the problem, feel free to ask them for a sample.
Best,
Ruben
Assays trying to measure total bacterial load are usually based on the amplification of universal segments of 16S rRNA genes. Previous assays were not adoptable to “direct” PCR protocols and/or they were not compatible with hydrolysis-based detection. Using the latest summary of universal 16S sequence motifs present in literature and testing our design with 500 liquid and 50 formed stool samples, we illustrate the performance characteristics of a new 16S qPCR assay which addresses well-known technical problems, including (a) positive priming reaction in the absence of intended target due to self-priming and/or mis-priming of unintended targets; (b) amplification bias due to non-optimal primer/probe coverage; and (c) too large amplicons for clinical qPCR. Stool swabs ranked into bins of different bacterial loads, show significant correlation with Ct values of our new assay. To best of our knowledge, this is the first description of qPCR assay measuring individual differences of total bacterial load present in human stool.
http://www.dmidjournal.com/article/S0732-8893(15)00126-1/abstract
- Badges
- Science method