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==Incorporation Mix== '''Required Materials''' '''*The volumes of the dNTPs in the various kits are specific to that kit''' '''*Do not mix and match dNTP vials form different cycle number kits''' *dNTPs: ff-dATP, ff-dCTP, ff-dGTP, ff-dTTP 2 each for the 36-cycle kit 1 each for the 18-cycle and 26-cycle kits *10X Incorporation Buffer v2.0 (5 ml) *dNTP Mix Diluent *SBS Polymerase 2 for the 36-cycle kit 1 for the 18-cycle and 26-cycle kits *Magnesium *Plastic Syringe *0.22 μm Cellulose Acetate Syringe Filter *50 ml Amber Tube *The volumes of dNTP Diluent, 10X Incorporation Buffer, and Magnesium vary depending on the sequencing kit you are using *Refer to the table below for correct volumes '''Reagents 18-Cycle 26-Cycle 36-Cycle''' dNTP Diluent 21ml 29ml 43ml 10X Incorporation Bufferv2.0 2.5ml 3.5ml 5ml Magnesium 150ul 210ul 300ul *Transfer the correct volume of dNTP Diluent to a 50 ml tube *Refer to the table of volumes for the sequencing kit you are using *Add the correct volume of 10X Incorporation Buffer v2.0 *Refer to the table of volumes for the sequencing kit you are using. *Vortex for 10 seconds. '''*NOTE Save the remaining 10X Buffer for the Incorporation Buffer''' *Pipette the contents of every ff-dNTP vial into the Incorporation Mix *Rinse each of the ff-dNTP tubes into the dNTP mix with 1 ml of Incorporation Mix *Invert several times to mix *Filter the dNTP mix through a 0.22 μm membrane filter *Transfer the mix into the supplied 50 ml amber tube labelled “dNTP Mix Tube.” *Place the dNTP mix on ice. *Remove the Magnesium solution from ice and allow it to thaw at room temperature for 10 minutes. *Vortex to mix. *Centrifuge to 10,000 xg for 2 minutes at 22°C. *Ensure that the vial contains no undissolved material or ice. *Add the correct volume of Magnesium solution to the dNTP mix on ice. *Refer to the table of volumes for the sequencing kit you are using. *Invert several times to mix and return to ice. *Centrifuge the SBS Polymerase to 10,000 xg for 1 minute at 22°C. '''*NOTE Two vials of SBS Polymerase are used in the 36-Cycle Kit. Only one vial of SBS Polymerase is used in the 18-Cycle and 26-Cycle Kits''' *Add the contents of each polymerase vial into the dNTP mix on ice. *Rinse each empty vial into the dNTPs mix three times with 1 ml dNTP mix *Record the weight of the reagent in the lab tracking worksheet. *Invert the dNTP mix several times to mix and immediately return to ice. *Keep on ice until ready to load.
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