Protocol tips
| Upstream tips |
Protocol tips |
Downstream tips |
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The microsatellite-enriched cDNA PCR products was digested with SalI and NotI using the following protocol: 20 μl (ca. 500 ng) cleaned cDNA, 3 μl 10 × SalI buffer, 1 μl (20 units) SalI (NEB) and 1 μl (10 units) NotI (NEB) at 37°C for 2 hours. |
After digestion, the cDNA was electrophoresed on 1% low melt gel (BIO-RAD). Fragments between 500 bp and 1200 bp were excised and cleaned using glassmilk (Gen 101). Approximately 50 ng cDNA was ligated to 25 ng pCMV-SPORT-6 vector (Invitrogen) which was used to transform XL-blue supercompetent cells (Stratagene). Schematic presentation of the method for microsatellite enrichment from normalized cDNA is shown in Figure Figure11. |
| Protocol tips |
| The microsatellite-enriched cDNA PCR products was digested with SalI and NotI using the following protocol: 20 μl (ca. 500 ng) cleaned cDNA, 3 μl 10 × SalI buffer, 1 μl (20 units) SalI (NEB) and 1 μl (10 units) NotI (NEB) at 37°C for 2 hours. |
| Downstream tips |
| After digestion, the cDNA was electrophoresed on 1% low melt gel (BIO-RAD). Fragments between 500 bp and 1200 bp were excised and cleaned using glassmilk (Gen 101). Approximately 50 ng cDNA was ligated to 25 ng pCMV-SPORT-6 vector (Invitrogen) which was used to transform XL-blue supercompetent cells (Stratagene). Schematic presentation of the method for microsatellite enrichment from normalized cDNA is shown in Figure Figure11. |
Publication protocol
The microsatellite-enriched cDNA PCR products was digested with SalI and NotI using the following protocol: 20 μl (ca. 500 ng) cleaned cDNA, 3 μl 10 × SalI buffer, 1 μl (20 units) SalI (NEB) and 1 μl (10 units) NotI (NEB) at 37°C for 2 hours. After digestion, the cDNA was electrophoresed on 1% low melt gel (BIO-RAD). Fragments between 500 bp and 1200 bp were excised and cleaned using glassmilk (Gen 101). Approximately 50 ng cDNA was ligated to 25 ng pCMV-SPORT-6 vector (Invitrogen) which was used to transform XL-blue supercompetent cells (Stratagene). Schematic presentation of the method for microsatellite enrichment from normalized cDNA is shown in Figure 1.
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