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aflp_454seq_for_pop_structure [2012/01/24 11:13] anniearchambault |
aflp_454seq_for_pop_structure [2012/02/02 15:51] (current) anniearchambault |
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| - | <WRAP box>{{:ligation_mix_.jpg|Ligation Mix}}<WRAP box>**Figure 1** Pictogram of the DNA fragments involved for ligating double stranded adaptors to DNA previously digested with EcoRI and MseI restriction enzymes, in the context of a modified AFLP method for genome complexity reduction.</WRAP></WRAP> | + | <WRAP box 800px>{{:ligation_mix_.jpg|Ligation Mix}}<WRAP box>**Figure 1** Pictogram of the DNA fragments involved for ligating double stranded adaptors to DNA previously digested with EcoRI and MseI restriction enzymes, in the context of a modified AFLP method for genome complexity reduction.</WRAP></WRAP> |
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| Digested DNA samples were amplified in a PCR reaction where the reverse primer is LibL_B_MseI_plus2 for all tubes, and the forward primer is specific to a population (Table 4). However, since the objective of the study was to reveal the genetic diversity at the population level rather than between each individual, the ten //Panax quinquefolius// samples for each population were all labeled with a same set of barcoded population-specific primers. Each sample was however amplified separately prior to pooling, to ensure an equimolar representation in the pool. Figure 2 illustrates the DNA fragments involved in the selective amplification step. | Digested DNA samples were amplified in a PCR reaction where the reverse primer is LibL_B_MseI_plus2 for all tubes, and the forward primer is specific to a population (Table 4). However, since the objective of the study was to reveal the genetic diversity at the population level rather than between each individual, the ten //Panax quinquefolius// samples for each population were all labeled with a same set of barcoded population-specific primers. Each sample was however amplified separately prior to pooling, to ensure an equimolar representation in the pool. Figure 2 illustrates the DNA fragments involved in the selective amplification step. | ||
| - | <WRAP box>{{:selective_amplification_mix.jpg?1000|Selective amplification pictogram}} | + | <WRAP box 800px>{{:selective_amplification_mix.jpg?700|Selective amplification pictogram}} |
| <WRAP box>**Figure 2** Pictogram of the DNA fragments involved in selective amplification of previously digested-ligated DNA, in the context of a modified AFLP method for genome complexity reduction coupled to multiplexing samples for high throughput sequencing. The selective primers therefore also contain a barcode (MID), and an instrument specific region (Libl-A and Key), in addition to the template specific region (EcoRI).</WRAP></WRAP> | <WRAP box>**Figure 2** Pictogram of the DNA fragments involved in selective amplification of previously digested-ligated DNA, in the context of a modified AFLP method for genome complexity reduction coupled to multiplexing samples for high throughput sequencing. The selective primers therefore also contain a barcode (MID), and an instrument specific region (Libl-A and Key), in addition to the template specific region (EcoRI).</WRAP></WRAP> | ||
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| <WRAP box 500px>**Table 5** Reaction mix for selective amplifications, in the context of a modified AFLP method for genome complexity reduction coupled to multiplexing samples for high throughput sequencing. | <WRAP box 500px>**Table 5** Reaction mix for selective amplifications, in the context of a modified AFLP method for genome complexity reduction coupled to multiplexing samples for high throughput sequencing. | ||
| ^ Reagent ^ Initial conc. ^ Qty added ^ Final conc. or Final qty ^ | ^ Reagent ^ Initial conc. ^ Qty added ^ Final conc. or Final qty ^ | ||
| - | ^ HF Buffer | 5X includes (15 mM MgCl2) | 6 µl | 1X | | + | ^ HF Buffer | 5X (includes 15 mM MgCl2) | 6 µl | 1X | |
| ^ MgCl2 | 50 mM | 0.6 µl | 2.5 mM | | ^ MgCl2 | 50 mM | 0.6 µl | 2.5 mM | | ||
| ^ dNTP | 10 mM | 0.6 µl | 200 µM | | ^ dNTP | 10 mM | 0.6 µl | 200 µM | | ||
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| - | <WRAP box 400px>{{:gel_amplif_12juillet2011.jpg?400|Gel picture}}**Figure 3** Electrophoresis of from a selective amplification of digested-ligated //Panax quinqefolius// total DNA with selective primers, which have a MID barcode tail, for a modified AFLP method for genome complexity reduction coupled to a high throughput sequencing. Lane 1: [[http://www.neb.ca/detail.php?id=N3014|Lambda BstEII ladder]]; Lane 2: ?</WRAP> | + | <WRAP box 500px>{{:amplify_digested_ligated_library.jpg?400|Gel picture for selective amplification of prepared library for AFLP-like method}}**Figure 3** Electrophoresis of the product of selective amplification of digested-ligated //Panax quinqefolius// total DNA with selective primers, which have a MID barcode tail, for a modified AFLP method for genome complexity reduction coupled to a high throughput sequencing. Two different MgCl2 concentrations were tested, and different temperatures for the primer annealing step. Lane 1: [[http://www.neb.ca/detail.php?id=N3014|Lambda BstEII ladder]]; Lane 2: 57 °C; Lane 3: 61.8 °C; Lane 4: 65.5; Lane 5: 68.7 °C; Lane 6: 57 °C; Lane 7: 61.8 °C; Lane 8: 65.5 °C; Lane 9: 68.7 °C</WRAP> |
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| <WRAP box 500px>**Table 6** Cycling conditions for selective amplifications, in the context of a modified AFLP method for genome complexity reduction coupled to multiplexing samples for high throughput sequencing. | <WRAP box 500px>**Table 6** Cycling conditions for selective amplifications, in the context of a modified AFLP method for genome complexity reduction coupled to multiplexing samples for high throughput sequencing. | ||
| - | ^ Step ^ Temperature (°C ) ^ Time ^ | + | ^ Step ^ Temperature (°C) ^ Time ^ |
| | Initial denaturation | 98 | 2 min | | | Initial denaturation | 98 | 2 min | | ||
| ^ 30 cycles ^ ^ ^ | ^ 30 cycles ^ ^ ^ | ||
