![](media/image1.jpeg){width="7.527777777777778in"
height="1.8333333333333333in"}

Evaluation of Genomic DNA Prepared for Cloning at the DOE Joint Genome
Institute

In this package, you have received the following items:

-   DNA Standard Kit (7 tubes):

1 Lambda HindIII Size Standard (Marker 2) -- 50ul

6 Lambda DNA Mass Standards -- 25ul of each

(15, 31, 63, 125, 250, 500 ng/ 5ul)

***Store kit at -20^0^C upon receipt.***

-   **Protocol for Genomic DNA QC Using Gel Electrophoresis**

Following your evaluation of the sample(s) on the QC gel**,** please
send an email to your project manager Kerrie Berry (<KWBarry@lbl.gov>),
or Lynne Goodwin (<lynneg@lanl.gov>), and Eileen Dalin
(<e_dalin@lbl.gov>) for review. Please include the following
information:

1.  An image of your gel saved as a TIFF file.

    a.  Please do not send jpg, pdf, compressed, or files containing
        more than 3 samples per pixel. The quantification software used
        at the JGI can't import gel images with the above file types.
        Also, if gels are overexposed or not ran the right distance, as
        indicated in the protocol, the software will have difficulties
        quantifying accurately.

2.  A well content description.

3.  The volume of sample(s) loaded.

4.  The dilution (if needed).

5.  The total volume of sample(s).

***The DNA should not be shipped until approval is received from your
Project Manager.***

Genomic DNA QC Using Gel Electrophoresis

> Version Number: 3.0
>
> Production Start Date: 11/16/2004
>
> Version 3.0 Date: 08/30/07
>
> Author(s): Eileen Dalin
>
> Reviewed/Revised by: Jan-Fang Cheng, Victor Dorsett

# Summary

Utilizing the concentration and size standards provided by the JGI, run
an agarose gel to evaluate the quality, quantity, and molecular weight
of your DNA sample(s).

# Materials & Reagents

+-------------------------------+--------------------+-----------------+
| **[Materials/Reag             | **[Ven             | **[Stock        |
| ents/Equipment]{.underline}** | dor]{.underline}** | Number          |
|                               |                    | ]{.underline}** |
+-------------------------------+--------------------+-----------------+
| *[Disposables]{.underline}*   |                    |                 |
+-------------------------------+--------------------+-----------------+
| Microcentrifuge tubes         |                    |                 |
+-------------------------------+--------------------+-----------------+
|                               |                    |                 |
+-------------------------------+--------------------+-----------------+
| *[Reagents]{.underline}*      |                    |                 |
+-------------------------------+--------------------+-----------------+
| Marker 2 Lambda HindIII Size  | MBI Fermentas      | SM0101          |
| Standard (\~50ng/ul)          |                    |                 |
+-------------------------------+--------------------+-----------------+
| DNA MassStandards (Lambda     | JGI (in house)     |                 |
| DNA)                          |                    |                 |
|                               |                    |                 |
| 15, 31, 63, 125, 250, 500ng / |                    |                 |
| 5ul                           |                    |                 |
+-------------------------------+--------------------+-----------------+
| GenePure LE Agarose           | ISC BioExpress     | E-3120-500      |
| (Generates)                   |                    |                 |
+-------------------------------+--------------------+-----------------+
| Ultra Pure Ethidium Bromide   | Invitrogen         | 15585011        |
| (10mg/ml)                     |                    |                 |
+-------------------------------+--------------------+-----------------+
| 1X Loading Dye                |                    |                 |
+-------------------------------+--------------------+-----------------+
| 50X TAE Buffer                | Invitrogen         | 24710-030       |
+-------------------------------+--------------------+-----------------+
|                               |                    |                 |
+-------------------------------+--------------------+-----------------+
| *[Equipment]{.underline}*     |                    |                 |
+-------------------------------+--------------------+-----------------+
| 12X14 Horizontal Device Comb  | CLP                | 75.1214-MT-25D  |
| (25 well 1.5mm)               |                    |                 |
+-------------------------------+--------------------+-----------------+
| 12X14cm Horizontal Gel        | CLP                | 75.1214         |
| Electrophoresis Device        |                    |                 |
+-------------------------------+--------------------+-----------------+

# Procedure

NOTE: All reagents/stock solutions should be prepared prior to the start
of the procedure.

1.  **[Gel & Sample Preparation]{.underline}**

    1.  Pour one \~100 ml 1% agarose gel containing 1X TAE and ethidium
        bromide (.15ug/ml). Use a narrow well comb.

    2.  Transfer 1ul of your genomic DNA sample(s) into clean tube(s).

        a.  Use more if the genomic DNA concentration is thought to be
            low \<50ng/ul.

        b.  Dilution of the genomic DNA might be required if the
            concentration is thought to be \>500ng/ul.

    3.  Bring the sample volume up to 5ul with 1X loading dye.

    4.  Mix & spin down sample tube(s).

2.  **[Gel Electrophoresis]{.underline}**

> *Note: Examples of gels displayed in Appendices 1 & 2*

1.  Loading of the gel:

    a.  Well 1 - 5ul of 15ng standard

    b.  Well 2 - 5ul of 31ng standard

    c.  Well 3 - 5ul of 63ng standard

    d.  Well 4 - 3ul of Marker 2

    e.  Well 5 -- 5ul of DNA sample

> *Note: If multiple samples are being run, load samples every other
> well and complete with loading f-i after the last sample.*

f.  Well 6 - 3ul of Marker 2

g.  Well 7 -- 5ul of 125ng standard

h.  Well 8 -- 5ul of 250ng standard

i.  Well 9 -- 5ul of 500ng standard

```{=html}
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2.  Run gel for \~40 min at \~120V in 1X TAE buffer. If a different
    electrophoresis set-up is being used, ensure the genomic DNA bands
    have ran ≥2 cm down from well and separation of marker is apparent.

3.  Remove gel from gel box and image. Save photo as a TIFF file.

4.  Analyze genomic DNA for molecular weight, quantity, and quality.
    Refer to the JGI Guidelines document to see the specific guidelines
    in the following areas for your genome type.

    a.  **MOLECULAR WEIGHT** (Pulse-field gel recommended to properly
        access size)

        i.  Refer to Marker 2 diagram on Appendix 1. Is the genomic DNA
            high molecular weight? Fosmid libraries require DNA \>40kb,
            check if DNA band is above the 23kb band.

    b.  **QUANTITY**

        i.  Compare genomic DNA band with standard bands (15, 31, 63,
            125, 250, 500ng) to obtain a concentration estimate. Then
            use the concentration estimate to calculate the total DNA
            available for this sample. The JGI will run the TIFF file
            through quantification software to determine the quantity of
            the DNA sample(s).

        ii. If Quantity One Software (Bio-Rad) is available, please
            refer to Appendix 2 for instructions.

    c.  **QUALITY**

        i.  How does the DNA look? Is the DNA a tight band or does it
            appear to be streaky, displaying signs of degrading and/or
            shearing? Is RNA present in your sample? A protocol to
            remove RNA from the sample can be located at
            <http://www.jgi.doe.gov/sequencing/collaborators/index.html>.

> *Note: Examples of good and bad DNA samples are displayed in Appendix
> 3*

# Reagent/Stock Preparation

**[6X Loading Dye]{.underline} [1X Loading Dye]{.underline}**

75ml 100% glycerol 400ul Nuclease-free H~2~0

125ml Nuclease free H~2~0 100ul 100% glycerol

0.05g Bromphenol Blue 100ul 6X loading dye

0.05g Xylene Cyanole FF

![](media/image2.png){width="1.3604166666666666in" height="2.75in"}

Appendix 2:

1\) Click on Quantity One Program Icon
![](media/image5.png){width="0.31666666666666665in" height="0.325in"}

2\) Open gel file image to be analyzed.

The following toolbar & gel image will be used for this SOP.

![](media/image6.png){width="3.872916666666667in"
height="0.30277777777777776in"}

![](media/image7.wmf)

3\) Click on the "Draw a box and expand the image inside" Icon
![](media/image8.png){width="0.2833333333333333in" height="0.2375in"}

Draw a box around first set of concentration standards to expand.

![](media/image9.png){width="4.371527777777778in"
height="0.9895833333333334in"}

4\) Click the "Contour" Icon
![](media/image10.png){width="0.23333333333333334in"
height="0.22083333333333333in"}. The following sub-Icon box appears.
![](media/image11.png){width="3.4743055555555555in"
height="0.3416666666666667in"}

5\) Click on the "Volume Contour Tool" Icon
![](media/image12.png){width="0.24097222222222223in"
height="0.22291666666666668in"}. Place cursor arrow on outer edge of
concentration standard, left click & HOLD. Slightly move cursor outward
until the contour's bounding outline completely encompasses the desired
band.

![](media/image13.wmf)
![](media/image14.png){width="2.4298611111111112in"
height="0.9881944444444445in"}

*For older versions of the software the contour tool is not available.
Use the "Volume Rectangle Tool" Icon*
![](media/image15.png){width="0.17430555555555555in"
height="0.17430555555555555in"} *instead.*

![](media/image17.png){width="1.2048611111111112in"
height="1.6840277777777777in"}

6\) Place cursor in center of the bounding area and double click. The
"Volume Properties" box will appear.

Make sure "Standard" is selected and input only the numerical value for
that particular concentration standard.

Click on the OK button. Repeat for the remaining concentration
standards.

![](media/image18.wmf)

7\) After all of the concentration standards have been assigned, repeat
Steps #5 & #6 for the unknown samples. Make sure "Unknown" is selected.
Remember when outlining the "Unknown samples" ONLY select the high
molecular weight section for analysis.

![](media/image19.png){width="5.175in" height="0.9027777777777778in"}

8\) Click on the "Display Volume Report" Icon in the sub-Icon toolbar
![](media/image20.png){width="0.13333333333333333in"
height="0.16666666666666666in"}.

![](media/image11.png){width="3.4743055555555555in"
height="0.3416666666666667in"}

9\) The following window will appear, make sure all of the appropriate
boxes are selected.

![](media/image21.png){width="3.4902777777777776in"
height="2.908333333333333in"}

10\) Click on the "Show Curve" button to display graphic plot of the
standers & samples. Standers are red plus symbols (+) and unknown
samples are blue triangles (∆). All value points should be in close
proximity to linear curve plot. The closer the "R-Squared" value *(at
the bottom of the page)* is to 1.0 the greater the accuracy. *For this
example the R-Squared = 0.983483*

![](media/image22.png){width="4.124305555555556in" height="2.65in"}

11\) Click on the "OK" button. Page returns to "Volume Report Options"
displayed in Step #9. Click the "Done" button on that page to see the
final report.

![](media/image23.png){width="4.124305555555556in" height="2.55in"}

12\) Determine the "**Concentration**" of each sample in "**ng/ul**".

Sample #1 = **188ng/ul**

Sample #2 = **246ng/ul.**

> *Note: If the sample volume loaded is greater than 1ul, divide the
> concentration estimate by the sample volume loaded.*

13\) Determine the total "**Quantity**" for each sample in "**ug**".

Sample #1) 188ng/ul \* 500ul (total volume of sample) = **94,000ng Total
(94ug)**

Sample #2) 246ug/ul \* 250ul (total volume of sample) = **61,500ng Total
(61.5ug)**

![](media/image25.png){width="1.6582677165354331in"
height="2.833464566929134in"}![](media/image26.png){width="1.3665354330708661in"
height="3.1110236220472443in"}![](media/image27.png){width="1.508267716535433in"
height="3.333464566929134in"}

![](media/image28.png){width="0.9366141732283465in"
height="3.0484251968503937in"}![](media/image29.png){width="1.9799212598425198in"
height="2.9925196850393703in"}![](media/image30.png){width="2.0956692913385826in"
height="3.0484251968503937in"}