Do you think I can make the product of 300bp to 400bp with SYBR Green? I know it is usually less than 200bp in various other purposes. Here I don't want to analyze the copy number, just want to see the amplification of genomic DNA. The PCR product will be further checked on an agarose gel to validate the amplification.
You can amplify even 300-400 bp with SybrGreen, but long amplicons may produce more than one mealt pick if the primers have low specificity, because amplicon could dissociates more than one time due to secondary structures (with different melt temperature). So, it depends from the specificity of your primers. Of course, the agarose gel will help you to confirm your expected amplification.
Although the optimal pcr product size recommended for SYBR green PCR reaction is 100-200bp, I have personally amplified upto 500bp without any problems. So I think you can go ahead with your experiment. Best of luck.
The amplicon length for SYBR green PCR assay is 100-150bp. But you can obtain sufficient amount of PCR Product with low concentration of the dye which ranges from 0.1-0.5 μM. If it is higher than this it cause inhibition of PCR
I did a SYBR green qRT-PCR with genomic DNA using the standard amplicon length of 150 bp and I could see bands of this size on the agarose gel afterwards.
qPCR amplicons are usually short as possible for maximum efficiency. Around 70 - 100bp is probably the best. If you plan to use these primers for conventional PCR and gel verification, go higher 120 - 200 bp, so that its distinctly identifiable from any primer-dimer bands.
Yes Arya, you can amplify 300-400 bp with SYBRGreen using genomic DNA if it does not require quantification.
You can amplify even 300-400 bp with SybrGreen, but long amplicons may produce more than one mealt pick if the primers have low specificity, because amplicon could dissociates more than one time due to secondary structures (with different melt temperature). So, it depends from the specificity of your primers. Of course, the agarose gel will help you to confirm your expected amplification.
For qRT-PCR, the apropiate amplicon length should be arranged between 80- 150 bp. However, amplicons up to 300 bp amplify well.
the best amplicon is 70-150bp to avoid any non-specific product through melting curve in addition to obtain high specificity
What my experience with real time PCR, particularly with SYBR green, it is intercalating Dye and generally keep the amplicon length between 100-150 bases. AS you increase the length the sensitivity or the amount of dye occupancy in amplicons goes to level beyond its plateau value and gives wrong results. And you get the CT also in earlier cycles as it is based on sybr green occupancy in amplicons. suggestions in such cases,we have to run a absolute number standards like cloned copy of gene together in separate well.
Many people use 150 base of target gene and 300 base of GAPDH gene, and papers also get published, in my opinion for sybr green assay, the difference of target and internal control standard has to be similar in length for relative quantitation.
The best size of SYBR Green Real-Time PCR to amplify genomic DNA is 200 to 300bp.
- Badges
- Science topic