Genotyping Glast- and Cx30-CreERT2 mice
Martine Perraut, Frank W. Pfrieger
(fw-pfrieger@gmx.de)
CNRS UPR 3212, University of Strasbourg,
Strasbourg, France
Original Publication
Slezak et al. (2007) GLIA
55:1565–1576
|
Products |
Ref |
Supplier |
|
DirectPCRLysis
Reagent |
102-T |
Viagen
Biotech/Euromedex |
|
Proteinase
K |
P6556 |
Sigma |
|
Failsafe PCR system |
035FS09901K |
Epicentre/Tebu-Bio |
|
Primers
(40 nmol) |
|
Eurogentec |
|
|
Primer |
Size
(bp) |
Sequence |
|
Cre |
TK139 |
~
350 |
5’-ATT-TGC-CTG-CAT-TAC-CGG-TC |
|
|
TK141 |
|
5’-ATC-AAC-GTT-TTG-TTT-TCG-GA |
|
Cont |
ADV28 |
~
250 |
5'-TTA-CGT-CCA-TCG-TGG-ACA-GC |
|
|
ADV30 |
|
5'-TGG-GCT-GGG-TGT-TAG-CCT-TA |
DNA Extraction
- Incubate tails overnight at 56°C in
DirectPCRLysis reagent plus Proteinase K (0.5 mg/ml) under constant agitation.
- Stop reaction at 85°C for 45 min.
- Spin for 1 min at 8000 rpm.
PCR reaction
- Note that the stock solutions
containing Cre primers (TK139/TK141) and primers for the positive control
(ADV28/29) should be at 100 µM. Store TK139/TK141 in small aliquots at -20°C.
ADV28/29 is stable at 4°C.
- The stock solution containing Taq is
at 2.5 U/µl.
- Prepare a reaction mix per tube/animal
according to the following table
|
Product |
Vol [µl] |
|
Primers (Cre + Cont, sense/antisense) |
0.08
each |
|
Taq |
0.80 |
|
Failsafe Pre-Mix |
12.50 |
|
H2O |
9.38 |
|
DNA |
2.00 |
- Use the
following program
|
1 |
2 min at 95°C |
|
2 |
30 sec at 95°C |
|
3 |
30 sec at 55°C |
|
4 |
30 sec at 72°C |
|
|
Repeat 30 times steps 2 - 4 |
|
5 |
10 min at 72°C |
- Separate PCR fragments by electrophoresis (1.5 % agarose)
for 45 min at 110 V.
Last Modification 15-Nov-2011