DNA Preparation Techniques

Routinely, the TMF uses distal toe clipping of mice between 7-10 days old as a method to identify genetically modified rodents and provide tissue for genotyping analysis. Distal toe clipping (up to the first knuckle) may be used with scientific justification in the approved animal-use protocol, but may only be used on neonatal animals. This collection should be performed before the animals are 10 days old unless they are inordinately small, at which point it can be delayed to 13 days of age. In any case, if an animal's eyes are open, appropriate anesthesia must be used.

For biopsy via tail clips, a tail sample of only 1-2 mm is needed when doing PCR, but about 5 mm is recommended for Southern blotting. The TMF uses isoflurane anesthesia for collection of a tail biopsy. This is humane and makes it easier to cut tails and mark animals by ear punch at the same time. Our protocol is as follows:

1. Induce anesthesia in the home cage by inserting tube from isoflurane vaporizer into water bottle opening. All procedures are done in a fume hood to avoid exposure of operator to isoflurane fumes.
2. While maintaining all animals under anesthesia, remove animals one at a time.
3. Wipe Xacto knife blade and heat in Bunsen burner flame to help cauterize wound and prevent cross-contamination.
4. Cut 1-2 mm of tail tissue with the Xacto knife and transfer with forceps to a labeled microfuge tube.
5. Identify animal with a unique ear-punch pattern.
6. Record sex and ID number of animal on tail-cutting record sheet.

Distal toe clipping has been endorsed by FELASA as being more humane than tail-clipping. An advantage of distal toe clipping is that it can be used both as a source of DNA and a method of identification. The only disadvantage is that it must be generally performed when the animal is  7-10 days of age. Ear punching can also serve both purposes, but it is somewhat more prone to cross-contamination of tissue samples.

Tissue samples should be stored at –20C if they are not going to be extracted immediately.

Two routes of blood collection that do not require anesthesia are the saphenous vein and the maxillary vein. One advantage of using blood as a source of DNA is that multiple samples can be collected from the same mouse, with very little trauma to the mouse. Tail-cutting is limited to two samples, while distal toe-clipping and ear-punching are limited to a single collection if they are also used for identification.

A good description, with pictures, of blood collection from the saphenous vein can be found here.

DNA Extraction

A variety of kits are available for extracting DNA from mouse tissue. These yield DNA suitable for most downstream applications, such as PCR, Southern blotting, sequencing, etc. Our facility has obtained good results with the Qiagen DNeasy Tissue Kit (catalog numbers 69504 or 69506), which has instructions specific for tail or toe tissue and blood.

We can also recommend two methods using homemade reagents, described below. These offer the advantages of lower cost and less hands-on time, but the DNA will not be quite as pure as that from the Qiagen kit. They both yield DNA suitable for PCR.

Proteinase K method:

• Final concentration of tail digestion buffer (TDB):
  • 50 mM KCl
  • 10 mM Tris-HCl (pH 9.0)
  • 0.1 % Triton X-100
  • 0.4 mg/ml Proteinase K
  1. Add 100 ul of TDB per tail piece (2-5 mm) in a microcentrifuge tube. Be careful not to cut too much tail.
  2. Incubate the tube at either 60 C for 3 hours, with gentle mixing every 30 minutes, or 55 C overnight.
  3. Incubate the tube at 94 C (or boil) for 10 minutes to denature the Proteinase K.
  4. Spin in microcentrifuge at top speed for 15 minutes. Use an aliquot of supernatant straight from the tube (e.g., 1 ul in a 25 ul reaction) for PCR. If the results are questionable, try a 1:5 or 1:10 dilution of the DNA.

HOTSHOT method

(G.E. Truett, et al, BioTechniques 29:52-54, July 2000)

1. Cut 1-2 mm tail and place in a 0.5 ml microfuge tube. Caution - larger pieces of tail can inhibit the PCR.
2. Add 75 µl Alkaline Lysis Reagent. Assure that the tail fragment is completely submerged.
3. Incubate at 95C for at least one hour (longer may be better – see below) and then store at 4C until you proceed to the next step. A thermocycler is convenient for this step.
4. Add 75 µl Neutralization Reagent using a new aerosol-barrier tip for each sample. Mix well, using tip to break up tissue. Some people like to centrifuge the tubes after this step and transfer the neutralized supernatant to a new tube, but this is not necessary.
5. When genotyping animals that are 6 weeks and older, we find that increasing the 95C incubation time to 2 hr yields better results. Preps made from tail pieces longer than 2mm may inhibit the PCR. Use 1 µl of neutralized supernatant per 20 µl PCR reaction.

Alkaline Lysis Reagent
to 25 ml water, add
62.5 µl of 10 N NaOH (final conc is 25 mM)
10.0 µl of 0.5 M disodium EDTA (final conc is 0.2 mM)
(pH should be about 12, but should not have to be adjusted)

Make fresh every 1-2 months. Keep solution at RT.

Note: because of the presence of EDTA in the alkaline lysis reagent, you may need to increase the amount of magnesium chloride in your PCR master mix. Your PCR conditions may need to be re-optimized when switching to this method of DNA preparation.

Neutralization Reagent
To 24 ml water, add 1 ml of 1 M Tris-HCl (final conc is 40 mM)
(pH should be about 5, but should not have to be adjusted)
Keep solution at RT.

Make 1 M Tris-HCl with Tris hydrochloride salt.

Plasmid DNA for Microinjection

One of the most critical steps in making transgenic mice is preparing the DNA for microinjection. Poorly prepared DNA can be toxic to the ovum and contaminants can clog the injection needle, which typically have inside diameters of 0.5 micron at the tip.

The TMF performs the final purification step for all plasmids submitted for microinjection. We require clients to submit a restriction digest of at least 50 micrograms of the plasmid, cut in such a way as to separate the transgene from as much of the plasmid backbone as is practical. This digest must be accompanied by a picture of a gel on which an aliquot of the digest has been run (with suitable MW standards), with the transgene band clearly indicated.

We will electrophorese the restriction digest on a preparative gel and purify the transgene fragment.

Bacterial Artificial Chromosome (BAC) DNA for microinjection

To receive the standard service guarantee, the TMF must isolate BAC DNA for microinjection. Clients should ship a stock of transformed bacteria to the TMF with information including the BAC size, the strain of bacterial host, and the antibiotic marker used to select for bacteria containing the BAC. BACs intended for microinjection can also be purified by the client, however in this case the standard service guarantee will not apply. The TMF analyzes all BAC DNA via PFGE prior to microinjection. Methods of preparing a BAC for microinjection into mouse eggs differ from those for smaller plasmids in several important respects. BAC preparations are easily contaminated with bacterial DNA or degraded DNA, either of which can significantly affect the yield of transgenic mice. Bacterial lysates must be handled very gently to avoid shearing of bacterial DNA (which makes it difficult to separate from the BAC DNA).

To prepare BAC DNA, we recommend use of the NucleoBond Xtra BAC DNA purification kit from Macherey-Nagel.

A detailed protocol for BAC DNA preparation can also be found at the University of Michigan Transgenic Core website.

BAC transgenic constructs can be microinjected as circular molecules. It is not necessary to remove the plasmid backbone. Although single-copy integration events may result in double-strand breaks within the transgene, most integrants consist of multiple copies. In these cases, recombination between individual BAC molecules apparently reconstitutes intact transgenes. See the following references:

1. Bishop JO. 1996. Chromosomal insertion of foreign DNA. Reprod Nutr Dev. 36:607-18
2. Brinster RL, Chen HY, Trumbauer ME, Senear AU, Warren R and Palmiter RD. 1985. Factors affecting the efficiency of introduction of foreign DNA into mice by microinjecting eggs. Proc. Natl. Acad. Sci. (USA) 82:4438-4442.
3. Camper, SA and Saunders, TL. 2000. "Transgenic rescue of mutant phenotypes using large DNA fragments." in: Genetic Manipulation of Receptor Expression and Function. Accili. D (ed.) John Wiley & Sons, New York. pp. 1-22.
4. Hammer RE, Krumlauf R, Camper SA, Brinster RL, Tilghman SM. 1987. Diversity of alpha-fetoprotein gene expression in mice is generated by a combination of separate enhancer elements. Science. 235:53-8.
5. Smith K. 2001. Theoretical mechanisms in targeted and random integration of transgene DNA. Reprod Nutr Dev. 41:465-85.

Plasmid DNA for Electroporation into ES Cells

Targeting constructs must be prepared by the client for electroporation using Qiagen EndoFree Plasmid Maxi Kit

We will linearize the plasmid DNA and perform the final purification prior to electroporation. At least 100 micrograms of supercoiled plasmid DNA must be supplied to our facility for electroporation. More DNA may be required if the client needs more than the usual number of colonies picked after growth under selective conditions.

We also need a gel picture of the plasmid, uncut and cut with the restriction endonuclease that we will use to linearize the plasmid, to demonstrate that the enzyme cuts only once.

Genomic DNA from ES Cells for Southern Blotting or PCR

Lysis buffer recipe:
10 mM Tris, pH 7.5
10 mM EDTA
10 mM NaCl
0.5% w/v Sarkosyl
sterile-filter for long-term storage
Just before use, add proteinase K (Sigma cat #P2308) to a final concentration of 1 mg/ml.

1. Starting with individual colonies picked into 96-well plates, split into duplicate sets of plates, and freeze one set for later recovery of positive colonies.
2. Continue to culture second set of plates until all or nearly all wells are confluent, changing media as needed. It does not matter if some wells are overgrown or start to differentiate.
3. When ready for DNA extraction, rinse wells twice with 1xPBS and add 50 ul of lysis buffer per well.
4. Incubate plates overnight at 56C in a humid atmosphere. We recommend putting the plates on several layers of wet paper towels in the bottom of a Tupperware container with a tight lid.
5. The next day, using a multi-channel pipet, add 100 ul per well of a mixture of NaCl and ethanol (150 ul of 5M NaCl mixed with 10 ml of cold absolute ethanol). Agitate the mixture while pipetting to prevent the NaCl from settling out.
6. Allow the plates to sit on the bench at room temp for at least 30 minutes (2 hours is better) without moving the plates. The DNA precipitates as a filamentous network.
7. Invert each plate carefully to decant the supernatant. The DNA should remain stuck to the wells. Blot the inverted plate against paper towels to remove excess liquid.
8. Rinse the precipitate 3 times by dripping 150 ul of 70% ethanol into each well with a multi-channel pipet. Decant the ethanol each time by inversion.
9. After the final wash, invert the plate and allow it to dry on paper towels on the bench. The DNA is now ready to be resuspended and cut with restriction enzyme.
10. If the DNA cannot be digested immediately, add 150 ul of 70% ethanol to each well, wrap the plates in Parafilm, and store them at 4C in a tightly closed container lined with wet paper towels.
11. Do not allow the ethanol to evaporate completely during storage. If storing for more than a week, decant the remaining ethanol and add fresh 70% ethanol. We do not recommend storing the plates for more than a week before cutting the DNA.