### **Sizing and Verification of DNA Clones**

> Version Number: x
>
> Start Production Date: x
>
> Stop Production Date:
>
> Author: Jason Olivas
>
> Reviewed by: x
>
> Signature:

# Summary

To size and verify DNA clones isolated at the Production Genomics
Facility.

# Materials & Reagents

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

##### [Disposables]{.underline}

> Pipette Tips
>
> 8-Strip Thin Wall PCR Tubes

##### [Stock Solutions]{.underline}

> Sterile Water
>
> 1x TAE
>
> 6x Loading Dye

##### 

##### [Reagents]{.underline}

> BstZ171 NEB R0594L
>
> NEB-3 Buffer
>
> BSA
>
> Lambda DNA-Mono Cut Marker NEB N3019L
>
> 1Kb Ladder Gibco 15615-024
>
> LE Agarose

##### 

##### [Equipment]{.underline}

> Biorad 1000 Mini-Chiller Biorad
>
> Biorad Pulse Field Gel Electrophoresis Box Biorad
>
> BioRad Variable Speed Pump Biorad

# Procedure

[Preparation for Digestion]{.underline}

1.  Obtain the DNA sample(s) to be sized and verified. The sample(s)
    should be at a concentration of approximately 30ng/ul.

2.  Aliquot 3uls of DNA and 5uls of TE into a PCR tray well. Repeat for
    the appropriate number of samples to be sized and verified.

3.  Seal and place the PCR tray in a deli refrigerator until needed.

4.  Make a cocktail of the following: 0.6uls of BstZ171/sample, 1.5uls
    of NEB-3/sample, 0.15uls of BSA/sample, and 3.75uls of Water/sample.

5.  Prepare the Mixed Standard if not prepared already.

> [Digestion Of DNA]{.underline}

1.  Add 5uls of the cocktail to each sample. You can take the total
    sample, divide by 8, and add the appropriate amount to an 8-strip
    thin-wall PCR tubes to make it easier to aliquot to several wells
    (if needed) using a Matrix pipette.

2.  Incubate overnight at 37 degrees Celsius in a PCR machine. Program
    should be in memory. If not, then set for 1 hour to cycle 24 times.
    After the 24 hour cycle, set for 1 cycle at 70 degrees Celsius for
    15 minutes. Final cycle should be at 4 degrees Celsius forever.

3.  When incubation is complete, add 5uls of 6x loading dye.

> [Preparation and Running of Gel]{.underline}

1.  Prepare a 100ml 0.8% agarose gel. Add ethidium bromide to the gel.

2.  Set up the pulse field gel electrophoresis (PFGE) tray so that the
    appropriate comb is used and the edges of the tray are tightly
    sealed.

3.  Pour all of the 100mls onto the gel tray and let it solidify.

4.  While the gel is solidifying, set up the PFGE box.

5.  Prepare 2L of 1x TBE. Pour into the PFGE box.

6.  Turn on the variable speed pump to 85. Turn on the Biorad 1000
    mini-chiller and set it to 14 degrees Celsius. This will eventually
    bring the temp of the 1x TBE buffer down to the desired temperature.
    Be sure the thermometer for the mini-chiller is inserted in the lid
    of the PFGE box.

7.  Gel should be solid and ready for loading.

8.  In the 1^st^ well, add 4uls of the Mixed Standard (Lambda DNA
    Monocut + 1Kb Ladder).

9.  Then load 9uls of the DNA sample you want run in the next lane.
    Repeat 8 times then add another standard lane. You should have 1
    Standard lane, 8 samples, and then another Standard lane. Standard
    lanes are lanes 1, 10, and 19.

10. Once the gel is loaded, close the box, and prepare the PFGE box to
    run.

11. Now enter these values in for the PFGE program: 14C for temperature,
    4.5V/cm for V Gradient, 120 degrees for Angle, Linear for Ramp,
    0.6sec for Initial pulse time, 7secs for final pulse time, and a Run
    Time of 13.5 hours.

12. De-stain with DI water for greater than 10 minutes.

> [Imaging, Sizing, and Verification of Digested Sample(s)]{.underline}

1.  Take a picture with a Fluor-S imager. Try and fill up the screen as
    much as possible. Focus and resolution are what are important here.
    Settings should be: Filter=Ethidium 520LP, Integration Time=90
    seconds, Light Source=Epi.UV, and High Resolution should be set
    already.

2.  With the gel photo taken, select the dotted rectangle icon (next to
    the arrow in the upper left corner of the frame) and lasso the
    portion of the image containing the gel lanes. Go to Analysis and
    select 'Auto Lane & Band Finding'. If the image is good the lanes
    will be neatly outlined. The bands called automatically must still
    be carefully examined for genuineness, and also for multiplicity if
    the results are to be useful.

3.  Go to profile analysis.

4.  Select Open Standard and select the 1Kb + Monocut standard.

5.  Make the Background type Valley-to-Valley.

6.  Set your standard lanes according to how the gel is loaded. Usually
    it will be 1, 10, and 19.

7.  The number (#) of lanes will depend on your gel. Enter the
    appropriate amount of lanes for each gel.

8.  MW should be kilobases (kb) and Mass should be micrograms (ug).

9.  Go to Profile Report and look at the Items to be Displayed. There
    should only be Name and Molecular Weight selected. If there are
    more, then highlight one and move it to the left.

10. Go to overlay settings and change the color of the standards to
    something other than what the lanes to be analyzed are.

11. Set the gel photo background to SPECTRUM, reverse to negative with
    the 'arrows in a circle' button, and adjust the contrast slider for
    dark blue bands against a light blue ground. This seems to give
    greatest sensitivity.

12. Edit bands using the image tools, as well as the profile window (the
    little mountain-range icon). After all this work, be sure to SAVE
    THE FILE.

13. Now once you have this done, go to View and select Profile Report.
    This should give you values that you will need to size and verify
    your clones.

14. With Profile Report window active, go to 'File' 'Export to Excel'
    and 'Active Window'. This will drop the values in an Excel sheet,
    where they can be easily manipulated. SAVE the Excel sheet as
    gel#.xls in E:/Isolations/Analysis.

15. Add up all the values for each clone. This is the estimated size of
    the clone base on what the computer program came up compared to your
    standards. Then compare the clone fragments from the digest to
    original clone digest to see if clones are the same. Pass/Fail of
    the clone depends more on the comparisons then the size.

16. If the clone fragments match up with the original digest of the
    clone, then the clone is said to be verified and can be passed on to
    the Subcloning group.

17. If it does not match up, then it is an incorrect clone and you need
    a new glycerol stock to proceed.

# Reagent/Stock Preparation

[0.8% LE Agarose]{.underline}

Weigh out 0.8g of agarose and add it to 100mls of 1x TAE. Heat until
boiling and the agarose is dissolved. Let solution cool before pouring
(usually between 50-55 degrees Celsius).

[6x Loading Dye]{.underline}

.5g Bromo Blue

.5g Xylene Cyan

75mls Glycerol

[175mls DD Water]{.underline}

=250mls of 6x Loading Dye

[Mixed Standard]{.underline} (Lambda DNA-Mono Cut Mixture + 1Kb Ladder)

200uls of Lambda DNA Mono-Cut Mixture (500ug/ml)

150uls of 1Kb Ladder (1000ug/ml)

750uls 6x Loading Dye

[1900uls of TE]{.underline}

=3.0mls of Mixed Standard