Guidelines for submission of Genomic DNA to the DOE Joint Genome
Institute

> The JGI is committed to successfully completing your project and from
> many years experience has learned that DNA quality (molecular weight
> and concentration) and quantity are two of the most critical
> components in the success of sequencing projects.
>
> **To aid in the success of your project, we mail you a Standards Kit &
> Protocol for performing the DNA gel QC procedure REQUIRED before
> submitting DNA to the JGI for sequencing.** The protocol, "Genomic DNA
> QC" will accompany the kit, or you may refer to it here:
> http://my.jgi.doe.gov/general/index.html
>
> The instructions that accompany the kit include a protocol for
> performing the DNA gel QC, examples of acceptable and unacceptable DNA
> preps, and the procedure for having your DNA approved for submission
> to the JGI.
>
> If you questions or concerns about JGI specifications for DNA, please
> contact jgi_project_management@lanl.gov and your project manager.

**[JGI Starting Material Specifications: Purity]{.underline}**

> *For prokaryotes, the starting culture should be axenic, strain pure,
> and started from a single colony or from a culture diluted to
> extinction.* It may not be possible to assemble a genome that is not
> derived from an axenic and strain pure culture. If an axenic and
> strain pure culture is not possible, description and justification is
> required.
>
> *For eukaryotes, the starting DNA should be free of contaminants
> and/or symbionts, should have \< 5% organelle DNA, and \< 1%
> polymorphism wherever possible. If a haploid form for the organism
> exists this is preferable.* It may not be possible to assemble a
> genome that is derived from a contaminated or polymorphic sample.

*Uncontaminated --* RNA blobs and discrete bands

> require clean-up. Please refer to recommended RNAse I clean-up
> protocol: http://my.jgi.doe.gov/general/index.html

**[JGI Starting Material Specifications: Quantity]{.underline}**

-   *75 -- 100 ug* of high molecular weight genomic DNA (HMW gDNA) for
    whole genome shotgun projects. If the organism's genome size is \>
    200MB, then 200 ug HMW gDNA is required.

> Quantity of gDNA should be measured by agarose gel using the DNA
> standards kit provided. Please refer to the "Genomic DNA QC" protocol:
> http://my.jgi.doe.gov/general/index.html

**[JGI Starting Material Specifications: Quality]{.underline}**

-   *High molecular weight* -- The bulk of the gDNA prep must be larger
    than the 23KB lambda Hind III marker on an agarose gel. In order to
    construct a fosmid library the majority of the gDNA should be
    \~40kb.

-   The concentration of resuspended gDNA must be \>= 100 ng / ul as
    judged by agarose gel / mass markers. See the "Genomic DNA QC"
    protocol for more information.

**[JGI Starting Material Specifications: Shipping]{.underline}**

> Prior to shipping samples to the JGI, the gDNA prep must first be
> reviewed & approved. Complete the appropriate RI
> (http://my.jgi.doe.gov/general/gettingstarted.html) and email it along
> with a well-labeled gel image of your gDNA prep to your project
> manager. See the "Genomic DNA QC" protocol for detailed instructions
> on running the QC gel or find the complete JGI protocol:
> http://my.jgi.doe.gov/general/index.html.
>
> Format for shipping in order of preference:

-   Resuspended in TE (10 mM Tris, ph 7.5 -- 8, EDTA 0.1 mM) at \>= 100
    ng/ul shipped on dry ice

-   Ethanol pellet shipped at RT or on wet ice. Note some ethanol
    pellets the JGI has received will not spin down and resuspend.

-   Dry pellet shipped at RT or on wet ice. Note most dry pellets the
    JGI has received will not resuspend.

> *DNA should be shipped to the JGI in **one tube**.* The tube should be
> legibly labeled with

-   Genus, species, strain,

-   Name of PI on Proposal,

-   Concentration,

-   prepped date, and

-   Name or initials of primary contact (primary contact indicated in
    "JGI Required Information" form).

> If the DNA prepared in multiple preps, each prep should be QC'ed
> separately, and good preps pooled into one tube for shipping to the
> JGI.
>
> **Please Note:** JGI procedures such as (CTAB) for isolating
> prokaryotic HMW gDNA and various eukaryotic HMW protocols are
> available on the following website:
> http://my.jgi.doe.gov/general/index.html
>
> Elution of genomic gDNA from Qiagen columns often yields gDNA that is
> sheared to a MW of \~ 20KB and is therefore not recommended for whole
> genome shotgun sequencing projects.
>
> Beware of poor quality enzymes as they are sometimes contaminated with
> genomic DNA from enzyme source microbes.

**Quality control-gel electrophoresis conditions:**

<http://my.jgi.doe.gov/general/index.html>

please see: "Genomic DNA QC.doc"

To determine MW, mass and concentration, run a 0.75 - 1% agarose gel at
1-3 v/cm for 3 hrs - overnight, using the narrowest comb available. Use
lambda / Hind III markers and mass standards (mass standards can be
lambda markers if they are quantified, although dilutions of uncut
lambda are probably best). The gDNA should be \> 23 KB lambda marker
with some gDNA held up in the well. Smearing below the 23 KB marker is
bad. RNA blobs are bad. Please see examples of passing & failed DNA
below *A picture of this gel should be emailed to the JGI contacts prior
to shipment of the DNA*. **A standards kit will be provided.**

You are ready for the next step:
*<http://my.jgi.doe.gov/general/gettingstarted.html>*

Step 3: Required Information Spreadsheets.
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