ExTerminator. Nucleotide terminators removal kit for cycle sequencing reactions.
Protocol cat# 444-50, 444-250. 01. A&A BIOTECHNOLOGY, Aleja Zwycięstwa ...
ExTerminator Nucleotide terminators removal kit for cycle sequencing reactions. cat. # 444-50, 444-250 Introduction
Protocol
The principle of ExTerminator nucleotide terminators removal kit is based on precipitation of cycle sequencing reaction products on the surface of especially designed membrane. The addition of Mix Blue reagent to the cycle sequencing reaction mixture enables an easy control of precipitation process. In the next step the binding-washing solution is added and the whole mixture is loaded onto Exterminator minicolumn. During the short centrifugation step the cycle sequencing products are bound by the minicolumn membrane while the excess of non-incorporated nucleotide terminators and sequencing primer pass through the membrane. The remaining salts and leftovers of other impurities are removed in the washing step. Subsequently the purified cycle sequencing reaction products are eluted directly by TSR (Template Suppression Reagent) and in case of capillary DNA sequencers samples are ready-to-denature without any additional steps needed (e.g. drying, dissolving in appropriate buffer).
Protocol 1. Add 5 µl of Mix Blue to cycle sequencing reaction mixture, performed in 10-20 µl volume. Note: If cycle sequencing reaction is less than 10 µl, add sterile water to reach the final volume of 10 µl.
2. 3. 4.
Add 100 µl of WP bind/wash solution and mix sample by pipetting. Load the whole sample onto minicolumn. Spin for 30 s at 12 000-14000 RPM.
5. 6. 7.
Apply on minicolumn 400 µl of WP bind/wash solution. Spin for 2 min at 12 000-14 000 RPM. Transfer the minicolumn to a new 1.5 ml tube (included) and:
Note: the light blue colour of minicolumn membrane is a result of efficient precipitation of sequencing products.
A. Capillary DNA sequencer: apply precisely onto the membrane 25 µl of sterile nuclease-free water nuclease-free or TSR (Template Suppression Reagent). B. Slab gel DNA sequencer: apply precisely onto the membrane 50 µl of sterile nuclease-free water.
8. Incubate for 2 min at room temp. 9. Spin for 1 min at 12 000-14 000 RPM. 10. A. Capillary DNA sequencer: Light blue colour of eluted sample confirms proper purification of sample which is ready for thermal denaturation directly in the tube with attached minicolumn. B. Slab gel DNA sequencer: Light blue colour of eluted sample confirms proper purification of sequencing sample. Dry out sample in vacuum dryer and dissolve in desired volume of loading buffer before denaturation. Note: Store the samples at -20 °C before analysis.
A&A BIOTECHNOLOGY, Aleja Zwycięstwa 96/98, 81-451 Gdynia, Poland tel. +48 58 6982194, fax +48 58 6228578, www.aabiot.com,
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