1) Cut toes of mice at 7-12 days of age and record sex, color, and strain. Place toes in marked, individual eppendorf tubes.

2) Add 0.1ml of lysis buffer to each tube. Incubate at 50oC for >3hrs or O/N.
Lysis Buffer: 100mM NaCl, 10mM Tris pH8.0, 25mM EDTA, 0.5% SDS,
add fresh 0.1mg/ml Proteinase K (100X, 10mg/ml in ddH2O stock)

3) Extract protein with phenol once and chloroform once. Precipitate DNA with 2 volumes 95% EtOH and spin 5min. Remove sup. and wash once with 70% EtOH. Quick spin tube, remove any extra liquid and add 0.5ml TE Buffer or ddH2O.

4) Prepare a cocktail of PCR reagents for each strain of mouse to be analyzed. For primer sequences click here.
Cocktail (for 10 reactions): 92.5ul ddH2O, 15ul 10X PCR Buffer, 15ul DMSO,
15ul 10mM dNTP, 5ul primer #1(10uM stock), 5ul primer #2 (10uM stock),
2.5ul homemade TAQ polymerase
10X PCR Buffer: 166mM Ammonium Sulfate, 670mM Tris pH8.8, 67mM MgCl2, 50mM 2-Me, 67uM EDTA
*Id3ko genotyping* – Add additional 3.1 ul of 50mM MgCl2 to PCR cocktail

5) Use 14ul cocktail + 1ul toe DNA for each PCR reaction

PCR program YZ1 :
1) 93^oC – 1.5 min (initial denature)
2) 93^oC – 0.5 min (denature)
3) 57^oC – 0.5 min (anneal)
4) 65^oC – 3 min (extend)
5) Goto step #2 39 times.
approximate running time is 3.5hrs with MJ thermal cycler.

NOTE: PCR reaction for Id3 ^flox:
H₂O — 10uL
10x PCR buffer — 1.5 uL
dNTP — 1.5 uL
DMSO — 1.5 uL
Primer (10uM) — 1 uL each
DNA — 1.5 uL
Use 18uL cocktail containing toe DNA for each PCR reaction and run using DAI program:
1) 94^oC – 1 min (initial denature)
2) 94^oC – 0.5 min (denature)
3) 55^oC – 0.5 min (anneal)
4) 72^oC – 0.45 min (extend)
5) Goto step #2 35 times.
6) 72^oC – 3 min

PCR reaction for samples marked ‘#’:
ddH₂O — 12.75 uL*
10x Bioline PCR buffer — 2 uL
dNTP — 0.4 uL
50 mM Bioline MgCl2 — 0.6 uL
Primer (10uM) — 1 uL each
Bioline Taq – 0.25 uL
Template DNA — 2 uL
Total — 20 uL
*If more than 2 primers are needed, deduct volume from ddH₂O
For PBase, home-made Taq can be used.

For Id2 GFP, ID2 ERCre, Rosa ^ZsGreen, PBase, Rosa ^FLPER and Rosa ^FLP1 (FLP1A+FLP1B) — use Id2ERCre program as described below.
For the remaining, including Rosa ^FLP1 (FLP1C+FLP1D), use DAI program.
Id2ERCre program:
1) 94^oC – 2 min (initial denature)
2) 94^oC – 0.5 min (denature)
3) 64^oC – 0.5 min (anneal)
4) 72^oC – 0.45 min (extend)
5) Goto step #2 35 times.
6) 4^oC – 0:00

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