A Simple Procedure for Isolating Tail DNA for Transgenic Analysis

Adapted from Miller et al. (1988 NAR ) "A Simple Salting Out Procedure for Extracting DNA from Human Nucleated Cells."

1. Place (totally submerge) tail biopsy (1.0 - 1.3 cm; make sure mouse is at least 2 weeks old, no older than 4 weeks) in 450ul Tail Lysis Buffer.*
   
   
2. Add 25ul 10 mg/ml proteinase K.
   
   
3. Incubate 55°, O/N.
   
   
4. Add 18ul 13 mg/ml RNase A.
   
   
5. Incubate 37°, 1 hr.
   
   
6. Add 160ul saturated NaCl (approx. 6M); shake vigorously (whole rack, by hand) for 15 sec. (Make sure the tubes are securely capped to prevent spills.)
   
   
7. Spin 15 min.
   
   
8. Pour supernatant into fresh tube, leaving pellet behind (sometimes the pellet can be very goopy and difficult to work with).
   
   
9. Add equal volume (about 500ul) isopropanol to the supernatant; mix gently by inversion to precipitate the DNA, which will come out of solution in visible "strings," followed by "clumping."
   
   
10. Wash the precipitated DNA "clump" 2x with 70% ethanol (about 1ml); 1x with 95% ethanol (about 0.5ml).**
    
    
11. Briefly spin (few sec.) residual ethanol after final wash to bottom of tube; remove the ethanol using drawn-out Pasteur pipet; air-dry the DNA briefly.
    
    
12. Add 100ul TE (pH 7.4) and allow the DNA to dissolve over several hours (usually requires 37°-55° for awhile).
    
    
13. For transgenic screening run 10ug DNA (cut with the appropriate restriction enzyme) in an agarose gel for Southern blotting or use for PCR analysis.
    
    

    *Tail Lysis Buffer:	50mm Tris-HCl (pH 8.0)
    	100mm EDTA
    	100mm NaCl
    	1%SDS

**Don't bother spinning the alcohol washes; just carefully pour off the isopropanol/ethanol leaving the DNA behind. It's a very good idea to pour off the alcohols into another tube (as opposed to down the drain!) in case you inadvertantly pour out the DNA. This way, you can still rescue your sample.

Transgenic Screening

For Southern analysis one needs a restriction digest that gives a unique size band(s) for the transgene or a probe that does not hybridize with endogenous mouse genomic sequences. Select an enzyme that will produce bands of predictable size. When choosing a restriction enzyme, keep in mind that in most cases the injected DNA fragment will integrate as a head-to-tail concatemer, or array, containing a few or several copies of the fragment. You might chose an enzyme that cuts the fragment used for microinjection 1x and use the fragment, itself, as the hybridization probe. Under these conditions, one will observe on the autorad either (i) a "fragment-length" band, the intensity of which will depend on the number of copies of the fragment integrated into the mouse genome, and two bands of unpredictable size that represent the junction fragments between the integrated transgene concatemer and mouse chromosomal DNA; or (ii) two bands of unpredictable size, representing the junction fragments between a single integrated transgene and mouse chromosomal DNA. Warning: The DNA can be readily digested with most enzymes; however, HindIII, XbaI, SalI and XhoI do not digest this DNA well.

Quantitation standards should also be used. For these standards use varying amounts of the fragment used for microinjection, coelectrophoresed with 10ug normal mouse carrier DNA (restriction enzyme cut). For founder-screening, amounts of fragment equivalent to 0, 1, 5, and 10 copies per genome ( DNA content is about 6 pg/diploid genome) are good values to start with. Remember to convert these numbers to those representing the number of copies that would be present in the 10ug of genomic DNA run on the gel. Note that the apparent copy number reflects both the actual copy number per diploid genome and the fraction of cells containing the foreign DNA. If a founder mouse is "mosaic" and some cells lack the transgene (i. e., transgene integration occured some time after the first embryonic cell division) the copy number will be underestimated. For this reason copy number should be confirmed in F1 transgenic progeny. NOTE: If your limit of detection is below 1 copy you will miss single-copy transgenics!

PCR may also be used for transgenic screening. Of course, the optimal conditions for PCRing the plasmid may be different than for the genomic transgene and may have to be worked out empirically. Also keep in mind the possibility of false negatives, especially when screening a limited number of potential founders. Furthermore, Southerns will still be needed for determination of transgene copy-number in F1 mice and for revealing differences in integration site between different lines. For these reasons you may want to consider PCR only for routine screening of mice once you get a transgenic line going and have worked out the conditions for PCRing the transgene out of genomic DNA. A few advantages of PCR are the short time needed to perform the analysis, the small amount of mouse tissue needed (e. g., an ear punch or drop of blood), and the young age at which the analysis can be performed (matter of days).