Transgenic Animal Facility

Phone:   265-2801/262-9495

Isolation and Purification of DNA Fragments for Microinjection

1. Restrict desired amount of DNA to completion (usually about 50µg digested overnight).
2. Test for completion by running small amount of minigel.
3. If digest is complete run prep size agarose gel.   This is usually 0.8-1.2% depending on the fragment size.   Use highly purified agarose such as Seakem GTG and use TAE buffer.   Do not include ethidium bromide in the gel.   Can run gel overnight for convenience.   Set up the outside lane with markers and the next lane with a small amount of sample.   Do not overload lanes with DNA.
4. After gel is run, cut lane with markers and next lane with sample away from rest of gel.   Stain with ethidium bromide and destain with water.   Photograph gel with ruler at edge; cut out desired band in sample lane and save gel as template.  Reposition this portion of gel next to remainder which contains the rest of sample.   Lay ruler on gel.   Using the cut out template and ruler as a guide, excise region of agarose which should have band from the remainder of the gel.
5. Electroelute DNA as directed in following protocol (page 2) changing ammonium acetate solution every 20 minutes for fragments < 1 kb and every 30-40 minutes for fragments > 1 kb.   Repeat 4 times.   After fourth time, stain and destain pieces of agarose to make sure DNA has been eluted.  
6. Precipitate eluted DNA with 2.5 volumes of ethanol: at least 1 hour at -20°C followed by microfuge spin for 20-30 minutes at 4°C.  
7. Resuspend pellets in 20µl TE buffer, pH=7.4 and combine aliquots into one or more tubes.   At this point run a minigel to make sure DNA has been recovered. 
8. Pass sample through elutip column as described in following procedure (pages 3-4), diluting sample to 1ml with low salt buffer as described.
9. After elutip procedure, precipitate one final time to concentrate.
10. Be sure to allow pellet to dry thoroughly, either on bench top or in speed vac.
11. Resuspend pellet in 50µl TLE (10mM Tris pH 7.5/0.1mM EDTA).
12. Quantitate fragment on minigel by running 0.5µl, 1µl of sample next to known quantities of a DNA fragment of approximately the same size (i.e. calculated amounts of fragments derived from an appropriate pBR322 digest, for example)

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Electroelution

 

1.   Fill chamber with buffer.

 

2.   Remove air bubbles from V-chambers with pasteur pipet which has the ends covered with tubing (to avoid scratching the apparatus).

 

3.   Preclean apparatus by electrophoresing at 100 V for 15 minutes.

 

4.   Dump out buffer, replace with new.

5.   Place gel slices into sample wells. Slices should fit size of wells. Orient slices as they were during electrophoresis in the gel.   Incubate 5 minutes.   Meanwhile, remove air bubbles from V-chambers and carefully place 100µl of 8M ammonium acetate with bromophenol blue into each well which is in use.   Use a drawn out pipetman tip to add salt solution.

6.   Electrophorese at 100 V for 20-40 minutes depending on the size fragment.   After allotted time, remove 100µl ammonium acetate solution from V-chamber with drawn out pipetman tip and place in eppendorf tube.   Remove two more 100µl aliquots from well and add to tube.   Add 2.5 volumes of ethanol to tube; store at -20°C.

7.    Add new 100µl aliquot of ammonium acetate solution to V-chamber.   Be careful not to contaminate stock solution with DNA from eluter (i.e. use new pipetman tip to add fresh solution).

8.    Repeat steps #6-8, three times or until all DNA is out of gel pieces.

9.    Return to main protocol (page 1);   step 6.

Notes:

10. Clean electroeluter .   Dump out buffer; replace with fresh buffer; remove air bubbles; electrophorese for 30-45 minutes.   Empty and wash with distilled water.

 

11. Theoretically, the eluter should now be ready for another run.   However, keep in mind that others may not be as conscientious as you.   Preelectrophoresing the unit (i.e. step 1) before you begin your fragment elution is wise.

12. Even with great care, it is possible to get cross-contamination of samples if more than one type of fragment is eluted together.   At the least, separate different fragments by one empty well.   To avoid this problem, of course, it is best to only work with one type of fragment at a time.

Buffer:      20mM Tris, pH 8.0/5mM NaCl/0.2mM EDTA

for 1 liter:         20 ml of 1M tris, pH 8.0

1 ml of 5M NaCl

0.4 ml of 0.5M EDTA

Page 3

Schleicher & Schuell

Keene, OH   03431

800-245-4024

ELUTIP-d

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Rapid Method for Purification and Concentration of DNA

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Conventional methods for the purification of DNA can be time-consuming and often can result in low recovery.   The S&S Elutip-d columns offer the researcher a RAPID alternative to conventional techniques, i.e partition into an organic phase, centrifugation, and absorption to a solid support.   In addition, the Elutip-d is designed to both concentrate and purify samples of DNA.

Key features of the Disposable Elutip-d are:

RAPID- DNA can be concentrated and purified in minutes.

BROAD RANGE -recovers DNA fragments from 50 bp 50,000 bp.

EFFICIENT -As high as 95% recovery of DNA over the range of 10 ng to 100 ng.

EFFECTIVE -Eliminates inhibitors to restriction endonuclease digestion.

How to use the Elutip-d:

The Elutip-d   column can be used directly to purify and concentrate dsDNA, if the DNA is already in a buffered suspension.   However, if the DNA fragments have been separated on a gel, the DNA must first be extracted from the gel before the Elutip-d column can be used.   Once the DNA has been extracted, the Elutip-d can be used to rapidly prepare the DNA sample.

Outlined below is a guide to selecting a method of extracting the DNA from the gel prior to using the Elutip-d column.

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                                                               DNA Extraction Methods

TYPE OF GEL     TYPE OF SAMPLE     ELECTRO-    ELUSION BY            GEL                         GEL

                                                    ELUTION     DIFFUSION   DISSOLUTION       COMPRESSION

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Polyacryl-          Low molecular               X                    X

amide                 weight DNA

                         High Molecular               X                                  X

                         weight DNA

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Agarose           Low molecular                X                    X

                        weight DNA

                       High molecular                 X                                 X                            X

                         weight DNA

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Once the DNA has been isolated from the gel, it should be in a low salt buffer (0.2 M NaCl, 20 mM tris HCl (pH 7.3-7.5), 1.0 mM EDTA).  Sample should be diluted to 1 ml with low salt buffer.  Then the Elutip-d column can be used to concentrate and purify up to 20 ml of the DNA salt solution in four easy steps as follow:

A.   Preparation of the column

B.   Priming of the column

C.   Binding of DNA

D.   Elution of DNA

PART A:  Preparation of column for Elutip-D

1.  Prepare the following salt solutions.

LOW SALT HIGH SALT

0.2 M NaCl 1.0 M NaCl               

20 mM tris HCl (pH 7.3-7.5) 20 mM tris HCl (pH 7.3-7.5)

1.0 mM EDTA 1.0 mM EDTA

2. Load a 3 or 5ml syringe with 1-2ml of the HIGH SALT solution.

3. Cut off the tip of the Elutip-d column as close to the white disc as possible and then remove the top protector cap and attach to the syringe.

4. Force the HIGH SALT solution into the column in order to pre-wash the matrix.  Caution:  The Elutip-d column is designed for the solution flow in one direction only:  from the wide end (Luer lock hub) through the tip.  A reverse solution flow may cause matrix loss.

PART B:   Priming of column

1.  Load a second syringe with 8ml of the LOW SALT solution.

2.  Remove the column from the first syringe and reattach to the second syringe with the LOW SALT solution.  Prime the column with the LOW SALT solution to ensure that the HIGH SALT solution is washed from the column.

PART C: Binding of DNA

1.  Disconnect the column from the syringe.  Discard the syringe.  Load a 1ml syringe with the DNA sample.

2.  SLOWLY force the sample through the column allowing time for the DNA to bind to the matrix of the column.

3.  Disconnect the column from the syringe.  Do Not Discard.

4.  Load the same syringe with 1ml of the LOW SALT solution.

5.  Reconnect the column to the syringe and wash any remaining DNA sample from the syringe.

6.  Remove the column from the syringe.

PART D: Elution of DNA

1.  Load a 1ml syringe with 0.4ml of the HIGH SALT solution and then reattach the column to the syringe.  A second elution with 0.4ml HIGH SALT solution is optional.

2.  Elute the DNA from the column with the HIGH SALT solution into a 1.5ml Eppendorf tube.

3.  The DNA can be further concentrated by alcohol precipitation (for efficient recovery, use twice as much alcohol as buffer on a vol/vol basis).  The DNA pellet can now be redissolved in the buffer of your choice (we prefer TLE-10 mM Tris-pH 7.5, 0.1 mM EDTA).

RESULTS

The DNA fragments are now in a highly concentrated and purified form and are ready for   further investigations such as: restriction enzyme analysis, cloning and other in vitro reactions. The Elutip-d column was used to concentrate 1 mg E. coli DNA in 3 ml of 0.2 M NaCl buffer with > 90% recovery.   Furthermore, 10 ng of the same DNA resulted in >80% recovery.