Hi Everyone, I used the one step EDC coupling method to attach the NH2 modified forward primers (with the C6-T8 spacer) to the carboxyl group on the Magnetic nanoparticles. I got the coupling efficiency around 60-70%. However, my primers detached from the MNPs during PCR reaction and I can’t detect the biotin labeled amplicon on the MNPs after magnetic separation. But I can detect the amplicon in the gel electrophoresis of supernatant (left over after magnetic separation of MNPs). I used the MES buffer (pH6.0) for the coupling reaction and prepared EDC freshly in MES buffer (concentration varied from 10 mg/ml to 100 mg/ml) but problem remained the same. Can anyone suggest how to stabilize the covalent bonding between MNPs and Primers?
Thank in Advance
Soe Paing Kyaw
Dear Soe,
Regarding your question, you may find the following literature useful:
Nanoparticles Affect PCR Primarily via Surface Interactions with PCR ...
https://www.ncbi.nlm.nih.gov/pubmed/26030087
by Y Bai - 2015 - Cited by 3 - Related articles
Jun 9, 2015 - Nanoparticles Affect PCR Primarily via Surface Interactions with PCR Components: Using Amino-Modified Silica-Coated Magnetic ...
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Magnetic Nanoparticles PCR Enzyme-Linked Gene Assay for ... - NCBI
https://www.ncbi.nlm.nih.gov/pubmed/26667895
by Y Manthawornsiri - 2016 - Cited by 2 - Related articles
Dec 14, 2015 - Magnetic Nanoparticles PCR Enzyme-Linked Gene Assay for Quantitative Detection of BCR/ABL Fusion Gene in Chronic Myelogenous ...
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High-throughput SNP detection based on PCR amplification on ... - NCBI
https://www.ncbi.nlm.nih.gov/pubmed/19768607
by N He - 2009 - Cited by 9 - Related articles
High-throughput SNP detection based on PCR amplification on magnetic nanoparticles using dual-color hybridization. He N(1), Li S, Liu H.
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Regulation of PCR efficiency with magnetic nanoparticles in a rotating ...
www.sciencedirect.com/science/article/pii/S0009261411002685
by T Higashi - 2011 - Cited by 5 - Related articles
Apr 20, 2011 - This paper reports on the regulation of PCR efficiency with superparamagnetic nanoparticles in a rotating magnetic field. The level of efficiency ...
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Effects of Superparamagnetic Nanoparticle Clusters on the ...
www.mdpi.com/2076-3417/2/2/303/pdf
by T Higashi - 2012 - Cited by 6 - Related articles
Apr 2, 2012 - particle clusters on PCR efficiency by estimating the structures of such clusters in PCR. It was found that superparamagnetic nanoparticles tend ...
During the changing temperature the particlesmay agregate. You should do test in eppendorf to check the influence of temperatute on your nanoparticles.
After that check the chemical bonds using Infrared spectra or UV spectra
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