DNA quality and purity for pyrosequencing experiments?

07 July 2018 1 2K Report

Is there a 260/280 ratio cutoff for DNA that is to be used for bisulfite conversion and pyrosequencing? How pure does the DNA have to be?

Abhijeet Singh

https://www.illumina.com/documents/products/appnotes/appnote-methylseq-wgbs.pdf

As per Illumina, there is no specific requirement for the DNA quality with respect to the bisulfite sequencing. Thus, DNA quality of A260/A280 should be 1.8-2.0 (which is the general quality requirement for DNA). Also, if possible, use qubit for the quantification, as nanodrop can give false quantification of the DNA.

Badges
Science topic

Enhanced Yellow Fluorescent Protein (Aequorea victoria) DNA sequence?

I am on the lookout for the Enhanced Yellow Fluorescent Protein (Aequorea victoria) DNA sequence. Does anyone know where I can find it? Thank you in advance

03 March 2021 3,568 1 View

Does anyone know HRM channel in a 2 plex QIAGEN Rotor- Gene Q qPCR can be used as a separate channel?

I am going to have 3 different probes in my qPCR work that I am going to do. But I realized that the machine we have in the lab is a Rotor-Gene Q 2plex HRM Platform, saying it has green, yellow,...

01 March 2021 8,544 1 View

Which cell line should we use to perform biological dosimetry experiments?

We are preparing some experiments based on irradiating cells under different conditions in order to evaluate the effects in terms of DNA damage, genetic expression, etc. As our project is...

01 March 2021 3,355 3 View

Calculating volume of DNA required from DNA concentration?

Hi I am a bit confused. They are asking me to find out the volume of DNA required in ul (a total of 30-100 ng for genomic DNA) from the DNA concentration in the nanodrop reading which was 404.8...

26 February 2021 5,029 2 View

Has anyone used the Illumina ITS1 primer sets?

Hi everyone, Illumina provides a list of primers to amplify with high taxonomic coverage the ITS1 region for further fungal sequencing, but I cannot find the exact amount necessary of each...

25 February 2021 6,969 3 View

Errors in DNA sequences?

I am trying to better understand the scope of DNA replication and sequencing errors, e.g. 1. I have seen similar error rates of 10e4 to 10e5 for cell & instrumental DNA replication,...

24 February 2021 4,397 3 View

What might be the reason for obtaining two bands in plant genomic DNA extraction?

I got a thin band and a thick band i guess it would be the genomic DNA and the 260/280 ratio is 3.

21 February 2021 6,523 6 View

I can detect my protein but the encoding DNA sequence has stop codons within. Should I ignore the stop codons?

I constructed a synthetic DNA library (scFv, VH-VL orientation) with a 3' reporter and 8x histag. I cloned and expressed this gene following which I performed an ELISA. The ELISA results suggested...

19 February 2021 7,006 5 View

No title

17 February 2021 4,217 3 View

How much amount of DNA (ligation mix) should I use for transformation?

Hi Fellow Scientists! I am trying to clone my 300 bp insert into a 6 kb vector. I run a few different ligation reactions using (1:3 vector: insert molar ratios) 10, 20,30, and 40 ng/ul vector...

15 February 2021 9,563 6 View