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) ----------see next page
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