Genotyping by PCR

Methods for Mouse Genotyping by PCR (protocol 1)

1. Preparation of genomic DNA from the mouse tail.

1) Obtain about 5 mm of the mouse tail and cut it symmetrically into two pieces.
Note: Too long tail can result in the inhibition of PCR because of increased impurity.
Put the cut tail into 500 ul lysis buffer 9see below) in a 1.5 ml microfuge tube, which should be
with a rubber ring to prevent leakage of the content. Without DNA degration, tails can be stored at
-80 centigrade even after standing at room temperature for a couple of hours.
2) Incubate at 65 degree centigrade with gentle shaking overnight. When a part of tail tissue remains
because of inactivation of Proteinase K by the high temperature, addition of more Proteinase K is
recommended to lyse the tail completely.
3)--This step is optional--
Detect the quality of the genomic DNA by 1.0% agarose gel electrophoresis. 10 ul of the lysate is
enough for the detection. The sample may not be suitable for the following PCR unless >4kb DNA
is detected.
4) Heat the lysates at 95 degree centigrade for 10 minutes in a PCR machine or by boiling to inactivate
Proteinase K completely.
5) Spin the tail lysate briefly before transferring to a PCR tube to exclude the tissue debris. Proceed
directly to PCR using the tail DNA lysate as a template at a volume rate of 1/10 as follows.

2. PCR reactions.

Contents of PCR mixture for wildtype/knockout allele screening:
5 ul tail DNA solution: spin briefly before transferring to a PCR tube to avoid contamination of debris.
1 ul 10 uM primers (each upper and lower primer)
5 ul 10x KOD dash DNA polymerase (from TOYOBO Co. LTD.,Japan)
5 ul 2.0 mM dNTPs
32 ul dd H2O
Total volume of 50 ul

We recently found that the final volume can be reduced to 25 ul without mineral oil application.

Sequences of PCR primers: should be designed according to your target gene.
Primers for detecting wild-type allele
Primers for detecting knock-out allele

Methods for Mouse Genotyping by PCR (protocol 2)

Transgenic Genotyping from Tail Biopsies
Harvard University--MCB Department / HSCI
Remove .5-1 cm of the tail and place in 1.5 ml Eppendorf tube. (Store at -20oC until ready to digest).
Digest in Lysis Buffer* + Proteinase K (to 200 ug/ml final conc.).
Incubate in 55oC water bath overnight. (Vortex 1x after 1-2 h).
Add .5 ml Phenol:Chloroform:Isoamyl alcohol (25:24:1) to each tube and vortex for 30 sec.
Spin at top speed in a microcentrifuge for 5 minutes.
Transfer upper (aqueous) phase to new tube; make sure no debris from the interface is transferred.
Add 1 ml of 100% EtOH.
Vortex briefly or shake. Stringy white precipitate (the genomic DNA) should now be visible.
Spin briefly (<1 min) just enough to get the DNA to cling to the plastic, and decant supernatant.
Wash with 1 ml of 70% EtOH.
Let air dry until the pellet becomes partially translucent, but do NOT over-dry, or the DNA will not go into solution any longer.
Redissolve the pellet in 100 ml TE, pH 8.0.
Check concentration, and calculate the total yield, which should be around 10 to 50 mg.
Use 100 ng for subsequent PCR analysis.
*Lysis Buffer:
10 mM Tris-HCl, pH 8.0
25 mM EDTA, pH 8.0
100 mM NaCl
0.5% SDS

DNA from Tail Biopsies

Genotyping Transgenic Rodents by PCR

Isolation of DNA from Mouse Tail Biopsies

Lac-Z Detection in Tail Biopsies

Preparation of Mouse Tail DNA for Dot Blots or PCR

Universal Mouse Genotyping Protocol Using PCR

beta globin Primers

lacZ Primers

neo Primers