DNA Quantification Protocol

> Version Number: 1.1
>
> Production Start Date: 11/06/01
>
> Version 1.1 Date: 08/25/04
>
> Authors: Anna Ustaszewska
>
> Reviewed/Revised by: Tijana Glavina

# Summary

Upon isolation of the large BAC, fosmid, or cosmid clones, the DNA is
quantified and diluted or concentrated to a target concentration of
40ng/μl.

# Materials & Reagents

+-------------------------------+--------------------+-----------------+
| **[Materials/Reag             | **[Ven             | **[Stock        |
| ents/Equipment]{.underline}** | dor]{.underline}** | Number          |
|                               |                    | ]{.underline}** |
| *[Disposables]{.underline}*   | USA Scientific     |                 |
|                               |                    | 1402-9708       |
| 96-well skirted PCR           | Millipore          |                 |
| Microplate                    |                    | 42412           |
|                               | \-                 |                 |
| Centricon YM-100              |                    | \-              |
|                               | New England        |                 |
| Weigh boats                   | Biolabs            | N3011L          |
|                               |                    |                 |
| *[Reagents]{.underline}*      | Roche              | 236250          |
|                               |                    |                 |
| Lamda DNA (500μg/ml)          | Gibco/Invitrogen   | 24710-030       |
|                               |                    |                 |
| DNA Molecular Weight Marker   | Ambion             | 9858            |
| II                            |                    |                 |
|                               | Gibco/Invitrogen   | 15510-027       |
| (Lamda DNA-Hind III digest)   |                    |                 |
|                               | Gibco/Invitrogen   | 15585-011       |
| 50X TAE buffer                |                    |                 |
|                               | Sigma -Aldrich     | B3269           |
| TE buffer                     |                    |                 |
|                               | Millipore Milli-Q  | \-              |
| UltraPure Agarose             | System             |                 |
|                               |                    | \-              |
| UltraPure Ethidium Bromide    | Eppendorf          |                 |
| (10mg/ml)                     |                    | \-              |
|                               | Vortex-Genie       |                 |
| Bromophenol Blue-Xylene       |                    | \-              |
| Cyanole Dye Solution          | Bio-Rad            |                 |
|                               |                    | \-              |
| Milli-Q Water                 | \-                 |                 |
|                               |                    | \-              |
| *[Equipment]{.underline}*     | \-                 |                 |
|                               |                    | \-              |
| Centrifuge 5417C              | \-                 |                 |
|                               |                    | \-              |
| Vortexer                      | \-                 |                 |
|                               |                    | \-              |
| Fluor-S MultiImager           | \-                 |                 |
|                               |                    |                 |
| Gel Pouring Apparatus (inc.   |                    |                 |
| gel box, casting tray)        |                    |                 |
|                               |                    |                 |
| Gel Combs 50 teeth, 8mm       |                    |                 |
|                               |                    |                 |
| Microwave                     |                    |                 |
|                               |                    |                 |
| Stir bars                     |                    |                 |
|                               |                    |                 |
| Thermometer                   |                    |                 |
+-------------------------------+--------------------+-----------------+

# Procedure

NOTE 1: All reagents/stock solutions should be prepared prior to the
start of the procedure.

NOTE 2: When working with ethidium bromide, always wear nitrile gloves.

**1. [Gel Casting]{.underline}**

1.  Place the gel-casting tray in the gel-pouring stand and lock it into
    position. Make sure the tray is leveled.

2.  Prepare 1.0% agarose gel solution containing ethidium bromide (Refer
    to the **Reagent/Stock Preparation** section).

3.  Place a mercury thermometer in the agarose gel solution while it
    continues stirring over the magnetic stir plate in order to monitor
    the temperature.

4.  When the temperature of the agarose gel solution falls to 65^o^C,
    pour a thin 1.0% agarose gel solution containing ethidium bromide
    into the casting tray.

> ***NOTE:** The thinner the gel, the better the resolution, but make
> sure that it is even across the gel.*

5.  Insert 2 or 4 clean combs (50 teeth/8mm) in the designated slots of
    the casting tray and allow the gel to set for 30 minutes.

6.  Remove the combs from gel, making sure not to tear the gel.

7.  Remove the gel and place it in the electrophoresis box oriented so
    that the row of wells are closest to the negative (cathode, black)
    end of the gel box and fill the box with 1X TAE buffer so that it
    covers the gel but does not go over fill line. (Use fresh buffer if
    needed, change every 3 days).

> ***NOTE 1:*** *If there is too much buffer in the gel box, the
> mobility of the DNA on the gel will be impeded.*
>
> ***NOTE 2:** Dump out leftover 80ml of agarose into gel disposal tray.
> Rinse off thermometer and bottle in the sink area.*

**2. [Sample Preparation]{.underline}**

> ***NOTE:** If quantification of samples is delayed for a couple of
> days, samples can be frozen at 80^o^C.*

1.  Thaw samples to be quantified and concentration standards on ice.
    Samples should be kept on ice.

2.  While the samples are thawing, go to the Bio-Rad 1 computer in Rm
    145 (the small room inside the picking room) and enter the Venonat
    database  **Libraries DB**  select **Print out Excel sheet for
    Concentration Gel**  select up to 25 Library names for the samples
    that will be run on the concentration gel  select **Run Report** 
    Save this Excel file to [**\\\\C:drive\\Library
    isolation\\centricon\\Keeper\\name
    concentration\\**](../../../../../../../..//C:drive/Library%20isolation/centricon/Keeper/name%20concentration/)using
    the date and gel number (e.g.: 01-09-04gel22).

> ***NOTE:** The gel numbers should all be in chronological order.*

3.  In the Excel file, enter in the gel number (refer to Quantification
    notebook), sample numbers for each library, and leave the BAC conc.
    column blank. *Refer to Table 1 in Appendix A.*

4.  Print a copy to guide you in organizing the order of your samples
    (step **2.5**). Arrange sample tubes in same order as excel.

5.  In a 96-well PCR plate (colored or clear), pipette **2μl** of
    loading dye in the wells that will have DNA loaded in them. Pipette
    on one side of the well rather than on the bottom. Use a plate base.

6.  Make a loading diagram.

7.  Arrange samples according to the loading diagram.

8.  Vortex sample briefly before pipetting. Pipette **2μl** of sample
    per well. Pipette on the opposite side of the well from the loading
    dye.

9.  Repeat step **2.8** with the concentration standards. Load 2 sets of
    concentration standards.

10. Perform a quick spin of the plate in the centrifuge by allowing the
    centrifuge to ramp up to 1,000 rpm and then pressing the stop
    button. Make sure the sample and dye are at the bottom of the well.
    Cover plate with clear seal. Store source samples in deli when
    finished loading.

**3. [Loading and Running the Gel]{.underline}**

> ***NOTE:** Refer to Tables 1 and 2 in Appendix A to determine how to
> load the gel.*

1.  Load **4μl** of each sample into the wells using a single-channel
    pipette.

    a.  Leave wells 1-3 empty.

    b.  Leave wells 4-11 empty (for standards, to be loaded last).

    c.  Leave one well empty (#12).

    d.  Load the sample DNA in wells 13 -- 50 depending on the number of
        samples you have to run. Refer to the Concentration Gel Excel
        sheet to determine the sample order.

    e.  Load the concentration standards in wells 4 -- 11.

    f.  Load another set of concentration standards in the same order on
        row 2 of the gel.

2.  Run the gel **5 minutes at 100V**.

3.  After the gel has finished running, carefully remove it and place in
    a tray.

4.  Wash the gel in Milli-Q water, drain, fill again with Milli-Q water
    and let it sit in Milli-Q water for **1 hour**.

> ***NOTE:** This is done in order to wash ethidium bromide from
> background.*

**4. [Imaging the Gel ]{.underline}**

1.  Take the gel to the Fluor-S MultiImager in Rm145.

2.  Wipe-down the Fluor-S MultiImager's platform with 70% ethanol using
    Kimwipes, not paper towels.

3.  On the computer desktop located next to the MultiImager, open the
    **MultiAnalyst** program on the computer desktop. *Refer to Figure 1
    in Appendix A.*

4.  Under the **Image Preview Display**, select **POSITION** and
    position the gel on the MultiImager.

5.  If the gel image is out of focus, select **FOCUS**.

6.  Select **PREVIEW**.

7.  If the gel image is centered and focused, select **OFF**.

8.  Make sure that the settings on the MultiAnalyst are as follows:
    ***Ethidium 520LP**, Integration: **Manual**, Integration Time:
    **2.0** secs, Light Source: **Scan: UV 302**,**#2** or **Scan: UV
    302, #1** (either one is ok) Scan Width: **250mm**, High Resolution
    on.*

9.  Click on **CAPTURE**. Make sure picture seems correct.

10. Save the gel image as the date and gel number (e.g.:
    01-09-04gel22.bif) in the following directory: [**\\\\C:\\Library
    isolation\\centricon**\\**Keeper\\**](../../../../../../../..//C:/Library%20isolation/centricon/Keeper/).
    Also, print a copy for your notebook.

**[Rerun Gel]{.underline}**

1.  Carefully transfer the analyzed gel back onto the gel tray and place
    the tray back into the gel box.

2.  Load **10μl** of Marker II (different standard) into 3 empty wells
    of the gel (the well before the standards, the well in between the
    standards and the samples of unknown concentration and the well
    after the samples).

3.  Run the gel for additional **45 minutes at 125V** (or until there is
    clear separation between the individual bands of the marker). Use
    manual timer.

> ***NOTE:** While the gel is running, you may continue onto the next
> section -- Quantifying DNA samples.*

4.  Image the gel again (Image 5 seconds, longer than before) and save
    the new gel image as the date and gel number (e.g.:
    01-09-04gel22longrun.bif) in the following directory:
    [**\\\\C:\\Library isolation\\centricon\\longer gel
    runs**](../../../../../../../..//C:/Library%20isolation/centricon/longer%20gel%20runs).
    *Refer to Figure 5 in Appendix A.*

5.  Print copy of the picture for the Quantification Gels binder.

> ***NOTE:** All the samples we process carry inserts between 40-200kb
> in size. The heaviest fragment of the marker is 23kb in size. This
> means that all of the samples, being significantly larger in size than
> the top band of the marker, should appear above the 23kb band on the
> gel. If a sample band has traveled down beyond the 23kb band, it may
> have a deletion. Bring this to the attention of your supervisor.*

6.  Wipe down the Imager with 70% ethanol and a Kimwipe.

**6. [Quantifying the DNA samples]{.underline}**

***NOTE:** Turn the lights off for ease of viewing.*

1.  Zoom into the concentration standards on the gel image. *Refer to
    Figure 2 in Appendix A.*

2.  Using the **red box** icon to the left of the window, create a box
    around the first concentration standard band.

3.  On the menu bar, select the *arrow* icon, then use the arrow cursor
    to select the box that was just drawn around the concentration
    standard band.

4.  Holding down the **Ctrl** key, click and drag a box copy to the next
    concentration standard band and repeat until all 8 standard bands
    have blue boxes around them.

> ***NOTE:** It is imperative that the boxes around each band occupy the
> same area. Otherwise, the standard concentration plot will not be
> accurate.*

5.  Holding down the **Ctrl** key, click and drag a box copy to the
    black portion of the gel to create 2 background blanks.

6.  Double-click on each standard and background box, and in the *volume
    properties* menu, pick the type *(standard, background, unknown*)
    and input the known concentrations.

7.  On the menu bar, select the **View Volume Report** icon, then select
    the middle **BK** (background, "local") icon. *Refer to Figure 3 in
    Appendix A*.

8.  On the menu bar, select the **Volume Standard Curve and Data** icon,
    and do the following based on the standard curve. *Refer to Figure 4
    in Appendix A*.

    a.  Delete bad standards (any that appear to be too high or too low
        compared to the others). Change remaining standards to be
        accurate.

    b.  If the concentration standard points do not lie on the best-fit
        line, move the blue boxes on the gel image up or down to adjust
        the curve points closer to the line. Make sure the DNA band is
        contained within the box. If you cannot adjust the point close
        to the curve and keep the band in the box, you should not use
        that standard.

**NOTE:** We will usually load two sets of standards on the gel, then
once we take a picture, we see which set looks better and then use that
set. We do this because standards are crucial and if they look bad you
have to redo the entire gel.

c.  If your standard curve is good, the correlation coefficient should
    be close to 1. Do not use the curve if your coefficient is less than
    0.90. Coefficient =0.95, deleted 50μg/μl std.

```{=html}
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9.  Once the concentration standard points lie on the best-fit line,
    copy and paste the same size red box around each sample band.

10. Copy the concentration numbers into the Excel sheet from step
    **2.2**. Print a copy of the updated Excel file. Put a "d" by dilute
    ones, put a "c" for centricon.

11. Remove the overlays (File  remove overlays) from the gel image and
    print a copy of the picture, standard curve, and volume report.
    Place these in the Quantification Gels binder.

12. Once all samples are in the 20-40ng/μl concentration range, update
    the database so that all can be passed on to the next step
    (shearing):

    a.  Go back to the *BAC conc.* column of the Excel file in step
        **2.2** and change all of the concentrations of samples, which
        were diluted, to 40ng/μl.

    b.  Resave the file as "text only" file to a temporary folder on
        Nassau on the Network.

        i.  Go to **FILE ** select **Save As ** select **My Network
            Places**  open **tmp on Nassau**.

> ii.       If prompted, type in your username and password.
>
> iii.      Save the file as **MM-DD-YY** in the **Text (Tab
> delimited)** format.

c.  i.  

    ii. 

    iii. 

    iv. Upload the file to the Venonat database:

    v.  In Venonat, go to the Libraries DB  click on **Concentration
        Upload File Form** (*enter username and password*) enter the
        file name from **5.14.**b

    vi. Click on **UPDATE**. Check shearing queue to make sure they made
        it.

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13. Adjust the sample concentration (400μl) to 40ng/μl by dilution with
    TE buffer (if concentration is above 40ng/μl) or centricon (if
    concentration is below 20ng/μl). Do not adjust samples that fall
    between 20-40ng/μl.

> ***NOTE:** To determine how much TE buffer to add for dilutions, use
> the dilution equation in Appendix A.*

14. If the sample concentration is below 20ng/μl, centricon the sample
    to remove excess buffer:

    a.  Centrifuge the entire sample through the centricon column for
        **20 minutes at 4,000rpm**.

    b.  Discard the collected buffer.

    c.  Reverse the column and collect the concentrated DNA by
        centrifuging for **2 minutes at 4,000rpm**.

    d.  Bring the total sample volume to **100μl** by adding TE.

    e.  Rerun the sample on the next quantification gel. If the sample
        falls within the correct concentration range on the second gel,
        pass it on to shearing. If the sample fails the second gel, fail
        it in the database. This sample will go back into the Isolations
        queue.

15. Quick spin the adjusted samples in the small centrifuge 5417C for
    **5 min at 12,000 rpm**.

16. Pipette **200μl** of the DNA into a fresh tube using p200 and pass
    to the subcloning group by placing the tubes in a box labeled
    "Shearing All Others" in the bottom shelf of the -80^o^C freezer 008
    in Functional Genomics Lab (Rm 459).

> ***NOTE:** Make sure you are pipetting from the top of the tube to
> prevent aliqouting any contaminants that may clog the hydroshear
> machine in the later steps.*

17. Any remaining DNA in the original tube can be reracked in the Rm139
    DNA freezer.

**6. [Gel Disposal]{.underline}**

1.  Place the gel containing ethidium bromide in the labeled white bin
    in Rm139 for disposal. Once the gel is completely dry, it can be
    disposed of into the regular trash.

2.  Dump the gel box's TAE buffer into the sink and wash the gel box
    with Milli-Q water.

3.  Wash the gel pouring apparatus and gel combs with Milli-Q water.
    Make sure that there is no residual gel left behind.

# Reagent/Stock Preparation

**[1X TAE Solution]{.underline}**

For 20L: Mix 400ml 50X TAE with 19.6L Milli-Q H~2~O

For 10L: Mix 200ml 50X TAE with 9.8L Milli-Q H~2~O

For 7.5L: Mix 150ml 50X TAE with 7.35L Milli-Q H~2~O

For 5L: Mix 100ml 50X TAE with 4.9L Milli-Q H~2~O

**[1.0% Agarose Solution]{.underline}**

NOTE: Always wear nitrile gloves when working with solutions or gels
containing ethidium bromide.

1.  Add **200ml** 1X TAE buffer to a 500-ml bottle.

2.  Add **2g** agarose. (1g/100ml, i.e. 1%) --Measure on scale with
    weigh boat.

3.  Swirl in bottle briefly. Heat in microwave for 2 minutes.

> ***NOTE:** If still in solid form, it will settle on bottom, reheat up
> to 2 min. watching to make sure it does not boil over.*

4.  Add **2μl** Ethidium Bromide (1μl/100ml) while stirring. Flush tip
    in solution.

5.  Carefully add an autoclaved magnetic stir bar to the solution and
    mix the solution over a stir plate until the liquid is clear. Stir
    until temperature comes down to \~65^o^C, use thermometer.

> ***NOTE:** Make sure you add the stir bar after heating the solution
> in the microwave.*

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

To a 500-ml bottle, add and mix the following:**[\
]{.underline}**174ml Milli-Q water\
0.5g Bromophenol Blue\
0.5g Xylene Cyanole

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

To a 1-L bottle, add and mix the following:

> 400ml Milli-Q water\
> 100ml 100% Glycerol\
> 100ml 6X Loading Dye

**[Lamda DNA Concentration Standards]{.underline}**

10ng/μl, 20ng/μl, 30ng/μl, 40ng/μl, 50ng/μl, 60ng/μl, 70ng/μl, 80ng/μl

**Lambda DNA (500μg/ml) Serial Dilutions**

  ------------------------- --------- --------- --------- --------- -------- -------- -------- -------- -------- -------- -------- --------
  **Desired                 **200**   **150**   **120**   **100**   **80**   **70**   **60**   **50**   **40**   **30**   **20**   **10**
  concentration(μg/ml)**                                                                                                           

  **Initial concentration   500       200       150       120       100      80       70       60       50       40       30       20
  to use(μg/ml)**                                                                                                                  

  **Quantity of initial     300       675       740       750       720      700      600      500      400      300      200      100
  standard\***                                                                                                                     

  **TE to add\***           450       225       185       150       180      100      100      100      100      100      100      100

  **Quantity of new         750       900       925       900       900      800      700      600      500      400      300      200
  standard made\***                                                                                                                

  **Final Quantity\***      75        160       175       180       200      200      200      200      200      200      200      200
  ------------------------- --------- --------- --------- --------- -------- -------- -------- -------- -------- -------- -------- --------

\* All units are in μl

# SOP Approval

  ----------------------- ----------------------- -----------------------
  **DEPARTMENT**          **APPROVED BY**         **DATE**

  **Lab Supervisor**                              

  **Research &                                    
  Development**                                   

  **Instrumentation**                             

  **QC**                                          

  **Purchasing**                                  

  **EH & S**                                      

  **Informatics**                                 

  **Seq Assessment &                              
  Analysis**                                      

  **Dept Head of Prod                             
  Seq**                                           
  ----------------------- ----------------------- -----------------------

# Appendix A

Table 1. Concentration Gel Excel File.

  ---------- -------------- ----------- ------- ------- ---------- --------
  Lib. Id    Project        Library     Gel     Well    BAC Conc.  

                                                                   

  25646      3578618        AHCG        248     1       50.35      d

  25647      3596143        AHCH        248     2       30.18      

  25648      3571467        AHCI        248     3       31.73      

  25649      3599772        AHCK        248     4       50.22      d

  25650      3609042        AHCN        248     5       30.82      

  25651      3599327        AHCO        248     6       42.69      d

  25652      3612140        AHCP        248     7       58.6       d

  25653      3601856        AHCS        248     8       52.52      d

  25654      3611680        AHCT        248     9       43.15      d

  25655      3627846        AHCU        248     10      46.61      d

  25656      3571926        AHCW        248     11      50.2       d

  25657      3602860        AHCX        248     12      42.06      d

  25658      3608629        AHCY        248     13      45.25      d

  25659      3581190        AHCZ        248     14      35.93      

  25660      3568588        AHFB        248     15      27.74      

  25661      3568543        AHFC        248     16      38.92      

  25662      3589661        AHFF        248     17      40.74      d

  25663      3571447        AHFG        248     18      46.29      d

  25664      3600663        AHFH        248     19      43.05      d

  25665      2988708        AHFI        248     20      49.48      d

  25666      2975047        AHFK        248     21      59.17      d

  25667      2954569        AHFN        248     22      50.94      d

  25668      2975730        AHFO        248     23      45.68      d

  25669      2984259        AHFP        248     24      54.3       d

  25670      2959921        AHFS        248     25      58.46      d

  25671      2973597        AHFT        248     26      50.64      d
  ---------- -------------- ----------- ------- ------- ---------- --------

Table 2. Loading the gel:

<table>
<colgroup>
<col style="width: 6%" />
<col style="width: 6%" />
<col style="width: 6%" />
<col style="width: 5%" />
<col style="width: 5%" />
<col style="width: 5%" />
<col style="width: 5%" />
<col style="width: 5%" />
<col style="width: 5%" />
<col style="width: 5%" />
<col style="width: 5%" />
<col style="width: 6%" />
<col style="width: 7%" />
<col style="width: 7%" />
<col style="width: 8%" />
<col style="width: 8%" />
</colgroup>
<tbody>
<tr class="odd">
<td><strong>1</strong></td>
<td><strong>2</strong></td>
<td><strong>3</strong></td>
<td><strong>4</strong></td>
<td><strong>5</strong></td>
<td><strong>6</strong></td>
<td><strong>7</strong></td>
<td><strong>8</strong></td>
<td><strong>9</strong></td>
<td><strong>10</strong></td>
<td><strong>11</strong></td>
<td><strong>12</strong></td>
<td><strong>13</strong></td>
<td><strong>14</strong></td>
<td><strong>15</strong></td>
<td><strong>16-50</strong></td>
</tr>
<tr class="even">
<td>empty</td>
<td>empty</td>
<td>empty</td>
<td>Std</td>
<td>Std</td>
<td>Std</td>
<td>Std</td>
<td>Std</td>
<td>Std</td>
<td>Std</td>
<td>Std</td>
<td>empty</td>
<td><p>Sample</p>
<p>1</p></td>
<td><p>Sample</p>
<p>2</p></td>
<td>Sample3</td>
<td>Samples</td>
</tr>
</tbody>
</table>

Figure 1. MultiAnalyst software image.

![](media/image1.png)

Figure 2. MultiAnalyst Software after the gel image has been captured.

![](media/image2.png)

Figure 3. Volume Report chart.

![](media/image3.png)

Figure 4. Concentration Standard Curve.

![](media/image4.png)

The coefficient should be as close to **1** as possible.

Figure 5. Longer gel run with Marker II.

![](media/image5.png)

**Sample DNA Dilution Equation:**

M~1~V~1~ = M~2~V~2~ where:

M~1~ = starting concentration of the sample

V~1~ = 400μl

M~2~ = target concentration 40ng/μl.

Solve for V~2.~

Subtract 400μl from the value you obtain for V~2~ and add this final
volume of TE buffer to the DNA sample.

# Appendix B

**[AUDIT TRACKING]{.underline}**

**6/03/04** -- Danielle Mihalkanin and Duane Kubischta performed an
audit between 2:00PM -- 6:00PM (6/03). The user was Anna Ustaszewska.
Changes incorporated on **8/25/04**.