Genescan 600 liz internal size standard

The sites of the UV photoproducts and the MNase digestion were analyzed by single-cycle primer extension using Taq DNA polymerase (New England BioLabs). Two primers (Primer A: 5′-GCTATGACCATGATTACGCCAAGC-3′, Primer B: 5′- AGGGTTTTCCCAGTCACGACGTT-3′) with 6-FAM labelling at 5′ end were used for the UV irradiated samples to analyze both strands of the 601 and 601.2.4 sequences. For the samples digested by MNase, the labeled primer A and primer B was used for the 601 and 601.2.4 sequences, respectively. Primer positions are shown in Supplementary Fig. 2. For the extension, 200–500 ng of the UV irradiated or the MNase digested DNA were added with 20 μl of final volume of an amplification mixture containing 0.2 mM of each dNTP, 0.2 μM of the end-labeled primer, and 0.025 units/μl of Taq polymerase in Taq standard buffer (New England BioLabs). The samples were then denatured for 5 min at 95 °C and subjected to extension (1 min at 55 °C for primer annealing and 8 min at 72 °C for extension) in a thermal cycler. After the primer extension reaction, the products were collected by ethanol precipitation. The samples were re-suspended in 10 μl of deionized formamide containing 0.25 μl of the GeneScan-600 LIZ internal size standard (Applied Biosystems) and separated by a capillary electrophoresis instrument (3500 genetic analyser, Applied Biosystems).

20 μl of nucleosomes at each step of assembly/disassembly were irradiated with 5-ns-long pulses of 266nm UV laser beam at a frequency of 10Hz for a period of 1sec (Quanta-Ray INDI-40-10 10 pulsed Nd: YAG). After irradiation, DNA fragments were purified by phenol-chloroform extraction and ethanol precipitation followed by primer extension using Taq polymerase (New England BioLabs). Two primers (A and B) with 6-FAM labelling at the 5’ end, were used to analyse the sites of the UV photoproducts of the 601 sequences (Primer: 5′-GCT-ATG-ACC- ATG-ATT-ACG-CCA-AGC-3′, Primer B: 5′- AGG-GTT-TTC-CCA-GTC-ACG-ACG-TT- 3′). For primer extension, 200-500ng of the UV irradiated DNA were mixed in 20μl of the final volume of an amplification mixture (Taq polymerase (0.5U), dNTPs (0.2mM), Taq polymerase buffer (1×), each primer (0.02mM) and distilled water). Primer extension consisted of denaturation at 95°C for 6min, primer annealing at 55°C for 1min, and extension at 72°C for 9min was carried out. The products were then concentrated by ethanol precipitation. All samples were re-suspended in a 10μl solution containing 9.75μl deionized formamide and 0.25μl GeneScan-600 LIZ internal size standard (Applied Biosystems). The mixture was denatured for 5min at 95°C and separated by capillary electrophoresis using a 3500 genetic analyser (Applied Biosystems).

A total of 15 microsatellite DNA markers (MSL1, MSL2, MSL3, MSL5, MSL6, MSL9—Kohlmann & Kersten (2008) (link); Svi-4, Svi-6, Svi-L7, Svi-L8, Svi-18—Wirth, Saint-Laurent & Bernatchez (1999) (link); Pfla-L3, Pfla-L8—Leclerc, Wirth & Bernatchez (2000) (link), Za138, Za199—Dubut et al. (2010) (link)) were used to genotype all individuals. The markers were amplified in three multiplex PCRs using NED, PET, VIC and FAM end-labeled primers (Table 1).
Amplifications were carried out in 20 µl reaction volume and polymerase chain reaction (PCR) was conducted using AmpliTaq Gold^® DNA Polymerase (Promega Corporation, Madison, WI, USA) with Buffer II (100 mM Tris–HCl, pH 8.3, 500 mM KCl). The final reaction conditions and temperature profiles of the multiplex reactions are presented in Table S2.
The PCR was carried out in a Px2 Thermal Cycler (Thermo Electron, Waltham, MA, USA). The products of the multiplex PCR reactions were pooled together and run against a Genescan 600 LIZ internal size standard (Applied Biosystems, Foster City, CA, USA) on a 3,500 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). After electrophoretic runs, PCR fragments were sized using GeneMapper version 4.0 software (Applied Biosystems, Foster City, CA, USA).