Genotyping
Isolation of DNA from Mouse Tail Biopsies
2. Add 600 microliters of TNES and 35 microliters Proteinase K (10 mg/ml).
3. Incubate overnight (8-24 hr) at 55°C.
4. Add 166.7 microliters 6M NaCl. Shake vigorously for 15 sec.
5. Microfuge (12,000-14,000 xg) for 5 min at room temp.
6. Remove supernatant to new tube and add 2 volumes cold 95% ethanol (EtOH).
7. Spool precipitated DNA with closed end capillary tube.
8. Rinse DNA pellet with 70% EtOH (dipping in a tube filled with 70% EtOH works well) and allow to air dry 5-10 min.
9. Resuspend in 100-500 microliters of TE (10mM Tris, pH 8.0, 1mM EDTA). Volume depends empirically on pellet size.
10. Heat at 65°C for 10 minutes to aid dissolution of DNA.
11. Quantitate DNA and store at 4°C until needed.
TNES
10 mM Tris, pH 7.5
400 mM NaCl
100 mM EDTA
0.6% SDS
“6M” NaCl
Saturated salt solution stored at 37°C
The reference for the tail DNA preparation is:
Miller SA. Dykes DD. Polesky HF. 1988. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Research. 16(3):1215.
Transgene Copy Standards
PCR screens must be designed to detect transgene DNA at the single copy level or 0.1 copy level..
Southern Blots analysis of transgenic mice need copy standards to estimate copy number.
Copy standards are prepared by mixing non-transgenic tail DNA with a known amount of transgene DNA is to produce transgene copy standards.
For PCR, these standards can be used to determine the sensitivy of the PCR assay. You must obtain single copy sensitivity in your PCR prior to transgene submission. When you test DNA from potentially transgenic founder mice, run your single copy PCR test sample to ensure that your PCR assay is sensitive enough to avoid false negatives.
Southern Blots are commonly used to determine transgene copy number and the number of integration sites in transgenic founder mice.
Download a pdf file illustrating Southern Blot analysis of transgenic founders.
Calculation of Copy Number Standards
Assumption: the Haploid content of a mammalian genome is 3 X 109 bp
Assumption: you have 2 micrograms of tail DNA available
Since the transgenic founder mice are hemizygous:
mass of transgene DNA = N bp transgene DNA
1 microgram genomic DNA 3 X 109 bp genomic DNA
Example: for a 5,480 bp transgene insert or plasmid
mass of transgene DNA = 5,480 bp cloned DNA or
1 micrograms genomic DNA 3 X 109 bp genomic DNA
mass of transgene DNA = (5,480 bp cloned DNA) X (1 µg genomic DNA) or
3 X 109 bp genomic DNA
mass of transgene DNA = 1.83 picograms per 1 ug genomic DNA
transgenic mice will be hemizygous for the transgene, not homozygous
1.83pg transgene should be added to 2 ug of genomic DNA
Thus, to prepare a 1 copy standard: add 1.83 pg of transgene DNA to 2 microgram tail DNA
0.1 copy 0.183 pg
10 copy 18.3 pg
50 copy 91.5 pg
100 copy 183 pg
For use as a transgene PCR standard, use 200 ng of the spiked tail DNA as a substrate in a 25 ul PCR reaction as described: genotyping transgenic mice.
For use in Southern blot analysis, digest the tail DNA as you would for Southern analysis, and add the transgene insert DNA (not the entire plasmid) just before you load your gel. Remember to reserve one lane for genomic DNA only with no spike. For an example of copy standards in Southern blots, refer to Camper SA. 1987. Research applications of transgenic mice. Biotechniques 5, 638-650.Click here for more review articles.
Beta Globin Primers
5′ CCA ATC TGC TCA CAC AGG ATA GAG AGG GCA GG 3′ 32 mer
5′ CCT TGA GGC TGT CCA AGT GAT TCA GGC CAT CG 3′ 32 mer
Size of expected amplification product is 494 base pairs.
Thermal cycler profile:
94°C 30 seconds
60°C 90 seconds
72°C 120 seconds
35 cycles
72°C 10 min
4°C Hold
Konkel DA, Tilghman SM, Leder P. 1978. The Sequence of the Chromosomal Mouse beta-globin Major Gene: Homologies in Capping, Splicing and Poly(A) Sites. Cell 15:1125-1132.
lacZ Primers
5' TTC ACT GGC CGT CGT TTT ACA ACG TCG TGA 3' 30mer
5' ATG TGA GCG AGT AAC AAC CCG TCG GAT TCT 3' 30mer
Size of expected amplification product is 364 bp.
Thermal cycler profile:
94°C 60 sec
72°CC 120 sec
30 cycles
72°C 10 min
4°C Hold
neo Primers
The primers recognize sequences within the neor cassette of gene targeting vectors.
5′ AGG ATC TCC TGT CAT CTC ACC TTG CTC CTG 3′ 30mer
5′ AAG AAC TCG TCA AGA AGG CGA TAG AAG GCG 3′ 30mer
Size of expected amplification product is 492 base pairs.
Thermal cycler profile:
94°C 30 sec
72°C 120 sec
30 cycles
72°C 10 min
4°C Hold
Rat Prolactin Primers
5′ GCT TCT GAG CAA TGA CAC CA 3′ 18 mer
5′ ATT CCA GGA GTG CAC CAA AC 3′ 18 mer
Size of expected amplification product is 391 base pairs.
Thermal cycler profile:
- 95°C for 45 seconds
- 95°C for 45 seconds
- 55°C for 1 minute
- 72°C for 30 seconds
- Go To #2 30 times
- 72°C for 10 minutes
- Hold at 4°C
Rat Prolactin Sequence Source (Genbank Accession Number AH002235):
Gubbins EJ, Maurer RA, Lagrimini M, Erwin CR, Donelson JE. Structure of the rat prolactin gene. J Biol Chem. 1980 Sep 25;255(18):8655-62.