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==Removal of biotin strand from adapter== ===Purpose=== *Removal of the strand with biotin group using magnetic beads, so that we can obtain the top and bottom portion of the PE_b_Met and PE_t_Met adapter. *Use 1mg of Dynabeads (100ul) to 10ug of ds DNA ===Material=== *Dynabeads M-280 Streptavidin_Invitrogen_Cat. no. 112.05D (10mg, 6-7*10^8 Dynabeads per ml) *2x Binding and Washing Buffer (B&W Buffer) -[10mM Tris-HCL (pH 7.5)] -[1mM EDTA] -[2M NACL] Prepare 10ml B&W Buffer {| {{table}} | ||ul |- | 5M NACL||4000 |- | 0.5M EDTA||20 |- | 1M Tris-HCL||100 |- | ddH2O||5880 |- | Total||10000 |- | |} ===Prepare Streptavidin Dynabeads=== *Take 100ul Dynabeads per reaction, place the tube on magnet and remove the supernatant when the solution becomes clear ~2 mins *Remove the tube from magnet *Add twice the volume of the beads of 1x binding buffer along the the inside of the tube where the beads are collected and resuspend *Place the tube on magnet and remove the liquid when the solution becomes clear *Wash the beads for a total of 3 times with the 1x binding buffer (1M NaCl, 10mM Tris-HCl pH 7.5, 1mM EDTA) *Resuspend the beads to 5ug/ul, 200ul of 2X B&W buffer ===Immobilization of Nucleic Acids=== *Add the 25ul primer to 25ul Dynabeads, incubate at RT for 15 mins in the Thermal Mixer. ===Binding to magnet and washing=== *Seperate the biotinylated primer with a magnet for 3 mins *Remove the liquid from the beads with a magnet. *Wash the coated beads 3 times with 1XB&W buffer ===Denature using NaOH=== *After the final wash, rinse the beads with 50ul 0.1M NaOH, incubate at RT for 1min. ===Neutralize Product=== *Separate the supernatant from the beads with a magnet, and transfer the supernatant (eluted primer) to 70ul 1M Tris-HCl, pH 7.5. *Purify the 120ul neutralized primer by ethanol precipitation with glyco blue, resuspend with ddh2o *Add 12 ul 3M sodium acetate and 300ul 100% ethanol with 1ul glycoblue *Freeze in -80C for 20 mins *Spin at 10,000rpm for 20min at 4C *discard supernatant *Add 500ul 75% etoh spin at 10,000 for 5 min at 4C *Discard supernatant *let dry in hood ~10min *Add 20ul to resolve adapter
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