Genomic DNA Molecular Weight QC using Pulsed-Field Gel Electrophoresis

> Version Number: 1.4
>
> Production Start Date: 05/03/07
>
> Version 1.3 Date: 08/03/07
>
> Author(s): Hope Tice, Eileen Dalin
>
> Reviewed/Revised by: Victor Dorsett, Jan-Fang Cheng

***PPE Requirements:***

***Safety glasses, lab coat, and nitrile gloves should be worn at all
times while performing this protocol. Additional safety equipment is
required at designated steps.***

# Summary

Genomic DNA received at the JGI is run on a pulsed-field gel to
determine the molecular weight of the sample. This information is
crucial for determining if the gDNA is suitable for constructing a
fosmid library. Majority of the DNA fragments must be \>=40kb in size to
pass this QC. To accurately QC genomic DNA, this protocol should be
performed after the Standard DNA QC gel which provides the quantity
information of the sample.

# Materials & Reagents

  ------------------------------------------------ -------------------------- -----------------------
  **[Materials/Reagents/Equipment]{.underline}**   **[Vendor]{.underline}**   **[Stock
                                                                              Number]{.underline}**

  *[Disposables]{.underline}*                                                 

  Rainin pipette tips                              Rainin                     \-

  0.6 microcentrifuge tubes, assorted              Continental Lab Products   3435.AS

                                                                              

  *[Reagents]{.underline}*                                                    

  10X TBE Buffer                                   Invitrogen                 15581-044

  Genepure LE Low Melting Agarose                  ISC BioExpress             E-3126-125

  T7 Control DNA (100ng/µl )                       Epicentre                  T7C0204

  MidRange PFG Marker I                            New England Biolabs        N3551S

  Ultra Pure Ethidium Bromide (10mg/ml)            Invitrogen                 15585011

  Marker 2 Lambda HindIII Size Standard            MBI Fermentas              SM0101
  (\~33ng/ul)                                                                 

  Low Range PFG Marker                             New England Biolabs        N0350S

                                                                              

  *[Equipment]{.underline}*                                                   

  Chef-DR III System                               Bio-Rad                    \-

  Rainin Pipettes                                  Rainin                     \-

  Bio-Rad Fluor-S Imager                           Bio-Rad                    \-

                                                                              
  ------------------------------------------------ -------------------------- -----------------------

Procedure

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

**1. [Size Fractionation/Gel Separation]{.underline}**

1.  Prepare pulsed-field gel apparatus by turning on the unit and
    changing the 1X TBE Buffer (2.4-2.6L).

2.  Set the following conditions:

+---------------------+------------------------------------------------+
| > **% Agarose**     | > 1.0% Low Melt Agarose (no EtBr)              |
+---------------------+------------------------------------------------+
| > **Buffer**        | > 1X TBE                                       |
+---------------------+------------------------------------------------+
| > **Temperature**   | > 14^o^C                                       |
+---------------------+------------------------------------------------+
| > **Voltage**       | > 6 V/cm                                       |
+---------------------+------------------------------------------------+
| > **Pulse**         | > initial 1.0 -- final 15.0 sec                |
+---------------------+------------------------------------------------+
| > **Run Time**      | > 17 hrs                                       |
+---------------------+------------------------------------------------+
| > **Angle**         | > 120^0^                                       |
+---------------------+------------------------------------------------+

3.  Prepare 250 ml of 1% Low Melt agarose with 1X TBE buffer in a flask.

4.  In a 4^o^C deli, pour 1% gel to the top of the gel casting tray
    using a narrow well comb and let solidify.

    a.  NOTE: You will need to keep a small amount of the low melt gel
        solution to seal the marker wells at a later step.

5.  Aliquot ≥ 100ng DNA sample(s) into a labeled microcentrifuge tube.

6.  Add 1x loading dye to a final volume of **5μl** (if sample is of low
    concentration, final volume can be increased to 10μl).

7.  Mix and spin down the sample(s).

8.  

9.  After the gel has solidified, load a slice (\~500ng) of the MidRange
    PFG Marker I or Low Range PFG Marker DNA into the first & last
    wells.

10. Seal the wells with low melt agarose **(\~50-100** **µl)**.

11. When the wells have set and the pulsed-field gel unit reaches
    14^0^C, place the gel in the box.

12. 

13. **10μl** a2 and **5μl** Load the entire sample(s) into the 1% Low
    Melting gel. Leave a small well in between each sample and marker.

14. Optional: Load 60-100ng of Marker 2 and/or T7 Marker in empty wells.

15. Run the gel overnight with above parameters.

**2. I[mage Gel ]{.underline}**

1.  Prepare the Ethidium Bromide (Carcinogen/Irritant) staining solution
    (**25** **µl** of EtBr into **500ml** of ddH~2~O).

2.  Add the solution to the staining box.

3.  Using a spatula, remove gel from pulsed-field platform and place in
    staining solution.

4.  Place the gel on a shaker, using a gentle setting, for 30 minutes to
    stain.

5.  After 30 minutes, drain the staining solution and replace it with
    **500ml** ddH~2~O.

6.  Place the box back on the shaker and destain the gel for at least 1
    hour at a gentle shaking setting.

7.  Take a photo of the gel with UV imager.

8.  Look for DNA to be in the 48.5kb size range. If DNA is below this
    band, it will not create a fosmid library. If DNA is present in the
    48.5kb range, it is large enough to create a fosmid library. The
    bulk of the DNA should be at this size with little to no smearing
    revealing degradation of sample.

**3. [Pulsed-Field Gel QC of Genomic DNA and MidRange PFG Marker I
Example:]{.underline}**

![](media/image1.png){width="1.4090551181102362in"
height="3.147244094488189in"}![](media/image2.png){width="4.9992125984251965in"
height="3.180708661417323in"}

Lane 1. MidRange Marker (NEB) 500 ng

Lane 2. T7 DNA Mass Marker 5 µl 125 ng (overloaded, suggest \~60 ng)

Lanes 3-10. Genomic DNA samples 1 µl of each (100-150ng)

The estimated amount of gDNA samples loaded in Lanes 3-10 are 40, 1000,
400, 111, 500, 28, 250, 70 ng.

Lanes 11-14. T7 DNA Mass Marker 150, 175, 200, 250 ng

Lane 15. Marker II (lambda cut with HindIII) 330 ng

# Reagent/Stock Preparation

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

500ml 10X TBE Buffer 125ml Nuclease free ddH~2~O

4500ml Milli-Q ddH~2~O 0.05g Bromophenol Blue

75ml 100% glycerol 0.05g Xylene Cyanole FF

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

400ml Nuclease-free ddH~2~O

100ml 100% glycerol

100ml 6X loading dye