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Old 01-14-2011, 08:12 AM   #1
mazinga
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Default Bacterial genomic DNA sample

Hi my name is Manuela and I work in the field of environmental microbiology. I need to prepare a bacterial genomic DNA sample to be sequenced by Illumina sequencing system. I need information about DNA quality, DNA concentration and DNA eluition solution. Could anyone be so kind to give me these information?

Thank you for your assistance

Manuela
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Old 01-14-2011, 09:06 AM   #2
DMO
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Buffer = TE (or water)
Quantity = 3ug ( you can get away with less than this if you need)
260/280 = >1.7
260/230 = >2
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Old 01-17-2011, 07:03 AM   #3
mazinga
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Thanks for your reply.
Is bacterial genomic DNA integrity important for the genome sequencing?
How can the shearing during DNA extraction affect sequencing?
I attach a gel image of my genomic DNA samples (the marker is 1Kb Abgene ladder).
Could you please tell me if these DNA samples can be used to safely sequence and assembly by Illumina technology?
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Old 01-17-2011, 07:27 AM   #4
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Having intact DNA is important in order to get even shearing across the genome, and thereby more even representation of the genome in your final sequence data. If you have degradation you need to account for it in your shearing times and intensities. I dont know the size of the genome you are dealing with, but the DNA appears to relatively intact.

What is the size of genome?

How do yo intend to shear your DNA? Or are you sending this out to a core?
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Old 01-17-2011, 08:17 AM   #5
mazinga
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I have no idea about the genome size but it could be about 5000 Kb and I’m sending the genomic DNA out in a sequencing center
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Old 01-18-2011, 12:53 PM   #6
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Your DNA looks good. It is largely intact. To estimate the amount you have, use your agarose gel or a double-strand specific assay, like pico green. RNA and even very small amounts of phenol can cause you to drastically over-estimate the amount of DNA you have if you use UV spec (nanodrop, etc.) Phenol can be ruled out if the A260/A270 is > 1.2. But RNA, degraded or not,

Contrary to popular belief, phenol, by itself, does not result in a low A260/A230 ratio. I have dissolved 1 ul of molten phenol (unequilibrated) in 1 ml of pure water. The resulting nanodrop trace gave an A260/A230 ratio of 2.67, A260/A280 of 2.15 and a "DNA" concentration of 542 ng/ul. All with no DNA of any sort present in the solution

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Old 01-28-2011, 08:37 AM   #7
mazinga
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Thank you Phillipfor you interest!
Could you please tell me if there is a possibility to over-estimate the amount of DNA in the sample with very small amount of RNA even if it is no possible to visualize any trace of RNA in the agarose gel (like the gel I attached)?
My protocol for the preparation of bacterial genomic DNA do not use any sort of phenolic compounds: do you think my genomic DNA samples may be affected by any sort of phenolic contamination?
The bacterial strain I’d like to sequence has got a 100 kb megaplasmid. It is possible to safely sequence this extrachromosomal DNA if this megaplasmid is in the same samples? Or it necessary to supply separately the chromosomal DNA and the extracromosomal one?
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Old 01-28-2011, 11:42 AM   #8
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Quote:
Originally Posted by mazinga View Post
Thank you Phillipfor you interest!
Could you please tell me if there is a possibility to over-estimate the amount of DNA in the sample with very small amount of RNA even if it is no possible to visualize any trace of RNA in the agarose gel (like the gel I attached)?
There absolutely is! Think about it: does degrading all the RNA in your sample down to mono-nucleotides change the UV absorbance of those nucleotides? Not much. But you will no longer be able to see them on a gel -- your matrix will not resolve down to 1 nucleotide and even if it did, your dye would probably not bind in the absence of double-stranded polynucleotides.

Genomic DNA preps are the ones you want to either use fluorimetry to assay or compare them to known molecular mass standards on a gel.

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Old 01-28-2011, 12:24 PM   #9
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You don't need to purify the megaplasmid away; everything will be sequenced together & then your assembly program will try to put it all back together correctly. In an ideal world, you'd get two DNA sequences back out (1 for chromosome, 1 for megaplasmid), but in the real world you will get a collection of contigs, some from each.
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Old 01-31-2011, 10:47 AM   #10
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Ah yes--the megaplasmid.
I agree with krobison, megaplasmids are rarely more than 1-2 copies/cell. Should not interfere.

On occasion I have seen samples of genomic DNA that were prepared in the same centrifuge bottles as a large (high copy) plasmid prep. The contamination was substantial.

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