Hi,
I need urgent help. This is my first time working on NGS samples and quantitating pcr products (not dna extracts per se).
I will be submitting my samples (long-pcr products) for NGS sequencing this week. However, I need to figure out how to quantitate my pcr product and combine/pool them in equimolar ratios. How do I do this if I do not use qPCR and Bioanalyzer?
I have 2-4 different pcr products for each species (20 species total). Each pcr product has different sets of primers that are overlapping. How do I combine them so that I end up with only 1 tube of sample for each species?
Do I just pour them in 1 bigger tube? e.g. if I have 25ul pcr, combining 2 of them will give me a 50ul total pcr product?
I was advised to run a gel and compare the brightness of the bands; and if they have the same brightness, it would be okay to just combine them. Is this okay? I am not confident in trusting that 2 or more samples for each species have/will have same brightness if I cannot measure them or no criteria other than brightness to look at. Are there other ways of quantitating pcr products?
How do I combine pcr products with equimolar ratios? How do I compute this?
Thank you.
I need urgent help. This is my first time working on NGS samples and quantitating pcr products (not dna extracts per se).
I will be submitting my samples (long-pcr products) for NGS sequencing this week. However, I need to figure out how to quantitate my pcr product and combine/pool them in equimolar ratios. How do I do this if I do not use qPCR and Bioanalyzer?
I have 2-4 different pcr products for each species (20 species total). Each pcr product has different sets of primers that are overlapping. How do I combine them so that I end up with only 1 tube of sample for each species?
Do I just pour them in 1 bigger tube? e.g. if I have 25ul pcr, combining 2 of them will give me a 50ul total pcr product?
I was advised to run a gel and compare the brightness of the bands; and if they have the same brightness, it would be okay to just combine them. Is this okay? I am not confident in trusting that 2 or more samples for each species have/will have same brightness if I cannot measure them or no criteria other than brightness to look at. Are there other ways of quantitating pcr products?
How do I combine pcr products with equimolar ratios? How do I compute this?
Thank you.
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