Difference between revisions of "Vinexin Genotyping"
Davebridges (Talk | contribs) (pasted in protocol from Vinexin genotyping paper) |
Davebridges (Talk | contribs) (Revised adding sections for Primers and PCR Program) |
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From [http://dx.doi.org/10.1016/j.yexcr.2010.03.019 Crucial role of vinexin for keratinocyte migration in vitro and epidermal wound healing in vivo.] | From [http://dx.doi.org/10.1016/j.yexcr.2010.03.019 Crucial role of vinexin for keratinocyte migration in vitro and epidermal wound healing in vivo.] | ||
| − | == | + | ==Primers== |
| + | *F1 AAGCTGAGCGCAGAGCTGGACAAGGACCTG | ||
| + | *R1 CCTGGAGTCTGCAGTTTCTAAGTCTCTCCC | ||
| + | *F2 TGCGAACTTTTCCGGAGGAGGTGGTGTCACTGG | ||
| + | *R2 TCCCTACCTGTCTCTCTCACTCACCTCCAC | ||
| + | |||
| + | ==PCR Program== | ||
| + | *94C for 3min | ||
| + | *25 Cycles of: | ||
| + | **94C for 25s | ||
| + | **65C for 25s | ||
| + | **72C for 150s | ||
| + | |||
| + | |||
| + | ==Protocol From Paper== | ||
The genotypes of mutant mice were determined by nested PCR and confirmed by Southern blot analysis of genomic DNA from tail biopsies. Briefly, tail samples were incubated in lysis buffer (10 mM Tris (pH8.0), 150 mM NaCl, 10 mM EDTA, 0.1% SDS and 1 mg/ml proteinase K) at 55 °C for 4 h, followed by purification with phenol/chloroform and precipitation with isopropyl alcohol. The first PCR for nest PCR was performed using primers (F1 AAGCTGAGCGCAGAGCTGGACAAGGACCTG, R1 CCTGGAGTCTGCAGTTTCTAAGTCTCTCCC) under the following amplification conditions: 94 °C for 3 min, 25 cycles of 94 °C for 25 s, 65 °C for 25 s, and 72 °C for 150 s. A primer set (F2 TGCGAACTTTTCCGGAGGAGGTGGTGTCACTGG and R2 TCCCTACCTGTCTCTCTCACTCACCTCCAC) was used for the second PCR under the same amplification conditions to detect the wild-type allele (1.5 kb) and targeted allele (2.5 kb). In some experiments, another primer set (F2 and R3 TGGGTGGAAACATTCCAGGCCTGGGTGAGAGG) was used to detect the targeted allele only. For Southern blotting, SalI/EcoRI-digested genomic DNA was probed with a 0.4-kb fragment immediately upstream of the 5′ arm (Supplemental Fig. S1A, S1B). | The genotypes of mutant mice were determined by nested PCR and confirmed by Southern blot analysis of genomic DNA from tail biopsies. Briefly, tail samples were incubated in lysis buffer (10 mM Tris (pH8.0), 150 mM NaCl, 10 mM EDTA, 0.1% SDS and 1 mg/ml proteinase K) at 55 °C for 4 h, followed by purification with phenol/chloroform and precipitation with isopropyl alcohol. The first PCR for nest PCR was performed using primers (F1 AAGCTGAGCGCAGAGCTGGACAAGGACCTG, R1 CCTGGAGTCTGCAGTTTCTAAGTCTCTCCC) under the following amplification conditions: 94 °C for 3 min, 25 cycles of 94 °C for 25 s, 65 °C for 25 s, and 72 °C for 150 s. A primer set (F2 TGCGAACTTTTCCGGAGGAGGTGGTGTCACTGG and R2 TCCCTACCTGTCTCTCTCACTCACCTCCAC) was used for the second PCR under the same amplification conditions to detect the wild-type allele (1.5 kb) and targeted allele (2.5 kb). In some experiments, another primer set (F2 and R3 TGGGTGGAAACATTCCAGGCCTGGGTGAGAGG) was used to detect the targeted allele only. For Southern blotting, SalI/EcoRI-digested genomic DNA was probed with a 0.4-kb fragment immediately upstream of the 5′ arm (Supplemental Fig. S1A, S1B). | ||
Revision as of 15:13, 6 August 2012
From Crucial role of vinexin for keratinocyte migration in vitro and epidermal wound healing in vivo.
Primers
- F1 AAGCTGAGCGCAGAGCTGGACAAGGACCTG
- R1 CCTGGAGTCTGCAGTTTCTAAGTCTCTCCC
- F2 TGCGAACTTTTCCGGAGGAGGTGGTGTCACTGG
- R2 TCCCTACCTGTCTCTCTCACTCACCTCCAC
PCR Program
- 94C for 3min
- 25 Cycles of:
- 94C for 25s
- 65C for 25s
- 72C for 150s
Protocol From Paper
The genotypes of mutant mice were determined by nested PCR and confirmed by Southern blot analysis of genomic DNA from tail biopsies. Briefly, tail samples were incubated in lysis buffer (10 mM Tris (pH8.0), 150 mM NaCl, 10 mM EDTA, 0.1% SDS and 1 mg/ml proteinase K) at 55 °C for 4 h, followed by purification with phenol/chloroform and precipitation with isopropyl alcohol. The first PCR for nest PCR was performed using primers (F1 AAGCTGAGCGCAGAGCTGGACAAGGACCTG, R1 CCTGGAGTCTGCAGTTTCTAAGTCTCTCCC) under the following amplification conditions: 94 °C for 3 min, 25 cycles of 94 °C for 25 s, 65 °C for 25 s, and 72 °C for 150 s. A primer set (F2 TGCGAACTTTTCCGGAGGAGGTGGTGTCACTGG and R2 TCCCTACCTGTCTCTCTCACTCACCTCCAC) was used for the second PCR under the same amplification conditions to detect the wild-type allele (1.5 kb) and targeted allele (2.5 kb). In some experiments, another primer set (F2 and R3 TGGGTGGAAACATTCCAGGCCTGGGTGAGAGG) was used to detect the targeted allele only. For Southern blotting, SalI/EcoRI-digested genomic DNA was probed with a 0.4-kb fragment immediately upstream of the 5′ arm (Supplemental Fig. S1A, S1B).
