SeqDirect PCR Cleaning Kit SeqDirect 96 PCR Cleaning Kit. High throughput cleaning of PCR reactions for fluorescent cycle sequencing

Transcription

1 SeqDirect PCR Cleaning Kit SeqDirect 96 PCR Cleaning Kit High throughput cleaning of PCR reactions for fluorescent cycle sequencing Revision # 9903/ k14

2 SeqDirect PCR Cleaning Kit SeqDirect 96 PCR Cleaning Kit High throughput cleaning of PCR reactions for fluorescent cycle sequencing Application Manual Revision # 9903/ k14 Product Description # Reactions Cat.No. SeqDirect PCR Cleaning kit SeqDirect 96 PCR Cleaning kit Any Questions? Call Technical Support at (800)

3 TABLE OF CONTENTS 1. Introduction Key features The SeqDirect Product Line SeqDirect PCR Cleaning kits SeqDirect 96 PCR Cleaning kit Description of the SeqDirect kit components Experimental Protocols and Recommendations Data Agarose gel of successfully sequenced PCR products purified with kit Sequencing electropherogram of a cleaned PCR product Sequencing electropherogram of an unpurified PCR product Equipment SeqDirect PCR Cleaning Protocol SeqDirect 96 Protocol Troubleshooting visit us on the web at

4 1. Introduction SeqDirect and SeqDirect 96 PCR Cleaning kits are designed to purify PCR products for fluorescent cycle sequencing using a 20 minute, two-temperature incubation that is amenable to high throughput processing. Templates applicable to the procedure include single-band and multiple-band PCR products if using specific nested primers. The SeqDirect PCR Cleaning Kit is offered in several sizes as a concentrate to be reconstituted before use. The SeqDirect 96 kit is offered in a 96-well format ready-to-use in any prep number. In the SeqDirect procedure cleaning of PCR products is efficiently achieved using a proprietary combination of enzymatic and non-enzymatic methods to remove/neutralize PCR primers, dntps, starting materials and PCR buffer components. Ideally, PCR products are cleaned immediately following the amplification reaction especially if the PCR primers are going to be used for sequencing. The SeqDirect and SeqDirect 96 PCR Cleaning kits purify and stabilize PCR products eliminating the requirement to sequence immediately. 1.1 Key Features 20 minute PCR cleanup: One tube/one step with minimal hands on time No vacuum filtration or washing steps 100% sample recovery Clean PCR Products are stabilized for later sequencing Compatible with high throughput robotic or manual methods Available in 96-well format ready-to-use in any prep number 2. The SeqDirect PCR Cleaning Product Line The SeqDirect and SeqDirect 96 PCR Cleaning kits contain all the relevant components to clean PCR products form solution for fluorescent cycle sequencing. The SeqDirect PCR Cleaning Kit is offered in several different reaction sizes as a concentrate to be reconstituted with the provided Buffer Mixx before processing (Cat.No to ). The SeqDirect 96 PCR Cleaning kit is offered in a 96-well format ready to use in any prep number. Any Questions? Call Technical Support at (800)

5 2.1 SeqDirect PCR Cleaning Kits Product Amount # Reactions Storage Cat.No. SeqDirect PCR Cleaning kit C SeqDirect Mixx 2x 2 µl 2x 8-20 C Buffer Mixx 50 µl -20 C SeqDirect PCR Cleaning kit C SeqDirect Mixx 4x 2 µl 4x 8-20 C Buffer Mixx 100 µl -20 C SeqDirect PCR Cleaning kit C SeqDirect Mixx 24 µl C Buffer Mixx 250 µl -20 C SeqDirect PCR Cleaning kit C SeqDirect Mixx 2x 120 µl 2x C Buffer Mixx 2x 1.3 ml -20 C SeqDirect PCR Cleaning kit C SeqDirect Mixx 10x 120 µl 10x C Buffer Mixx 10x 1.3 ml -20 C 2.2 SeqDirect 96 PCR Cleaning Kit Product Amount # Reactions Storage Cat.No. SeqDirect 96 PCR Cleaning kit C SeqDirect 96 Plate 2 µl/well* 1-20 C SeqDirect 96 Seal 1 room temp to -20 C *ready-to-use cleaning/stabilizing reagents in 96-well PCR plate 2.3 Description of the SeqDirect PCR Cleaning kit components -SeqDirect Mixx: Proprietary mixture of enzymatic and non-enzymatic reagents to remove/neutralize primers, dntps, PCR buffer components and starting materials. It also contains inert stabilizers for product stability at room temperature upon drying in a vacuum centrifuge. -Buffer Mixx: Proprietary buffer mixture used to reconstitute SeqDirect Mixx for optimal activity. 6 visit us on the web at

6 3. Experimental Protocols and Recommendations 3.1 Data Agarose gel of successfully sequenced PCR products purified with SeqDirect PCR Cleaning kit. 5 µl of different PCR products run on 1% agarose gel prior to treatment with SeqDirect PCR Mixx. Lanes 1 and 8: 4 µl Mass ladder (2Kb = 200 ng; 1.2 KB = 120 ng; 600 bp = 40 ng; 400 bp is not visible; GIBCO BRL) Lane 2: 491 bp human lamin C lane 3: 650 bp GST Lane 4: 780 bp mouse P53 Lane 5: 510 bp yeast PHO5 promoter Lane 6 and 7: 1 KB and 700 bp yeast actin, respectively Sequencing electropherogram of a cleaned PCR product Sequencing electropherogram of unpurified PCR product Electropherograms for the mouse P bp fragment (see image 3.1.1, lane 4) sequenced with the forward PCR primer using Big dye chemistry (Perkin Elmer). The reaction was cleaned with Sephadex G50 (Pharmacia) and run on an ABI sequencer. Image after purification; image before purification. Any Questions? Call Technical Support at (800)

7 3.2 Equipment required for the SeqDirect and SeqDirect 96 PCR Cleaning protocols Thermocycler or heating block PCR tubes, PCR strip tubes or 96/384 well PCR Plates (SeqDirect Kit only) Multi-channel pipet or liquid transfer workstation for high throughput processing 96-well plate centrifuge (SeqDirect 96 only) 3.3 SeqDirect Protocol 1. Reconstitute the SeqDirect Mixx with Buffer Mixx before initial use and continue with step 2. To reconstitute, pulse spin to collect the SeqDirect Mixx at the bottom of the tube, add Buffer Mixx as per Table 1 and invert or tap with finger a few times to completely mix. Keep the reconstituted Mixx on ice when in use and store at 20 C. Table 1 Cat# # ReactionsSeqDirect Mixx Buffer Mixx µl 18 µl µl 216 µl µl 1080 µl The reconstituted SeqDirect Mixx is stable at -20 C for ~ 1 month with several freeze-thaw cycles. 2. Transfer 8 µl of PCR reactions to PCR tubes, tube strips, or 96/384 well plates. Note: If PCR amplifying directly from whole cells or tissues, spin PCR reactions for 5 minutes to pellet the debris before transferring 8 µl of the clear supernate. 3. Add 2 µl of reconstituted SeqDirect Mixx per tube/well and incubate for 5 minutes at 37 C. Incubate an additional 15 minutes at 72 C. Note: The two temperatures of this incubation step can be programmed into PCR thermocyclers. Do not use the heated lid option if available. 4. After the 72 C incubation, the PCR products are clean and ready for sequencing. -If sequencing is to be done on the same day, store the samples at 4 C until used. Follow sequencing guidelines below. -If sequencing will be at a later time, stabilize the templates by drying in a vacuum centrifuge at room temperature. Prior to sequencing add 10 µl ddh 2 O and spin for 10 minutes at room temperature. Follow sequencing guidelines below. 8 visit us on the web at

8 Note: the presence of inert stabilizers, which are activated upon drying, ensures optimal sequencing results for up to two weeks if the cleaned/stabilized PCR templates are stored at room temperature and several months if stored at 4 C or -20 C. Guidelines for sequencing reactions Amount of PCR products to use per sequencing reaction for the various sequencing machines. Use the same quantity for full and 1 /2 reactions. PE Applied Biosystems Beckman ABI 373 ABI 377 ABI 3700 CEQ µl 1-2 µl 1-2 µl 1-2 µl 3.4 SeqDirect 96 Protocol Note: The SeqDirect 96 plate containing cleaning/stabilizing reagents can be cut to any number preps immediately after removing from -20 C storage with the SeqDirect 96 seal on. Return the unused portion to -20 C. 1. Spin the sealed plate at 1000 rpm for 5 seconds to collect reagents at the bottom of the wells. Remove the seal and add 8 µl of PCR reactions per well. Cover plate with a new SeqDirect 96 Seal cut to size. Note: If PCR amplifying directly from whole cells or tissues, spin PCR reactions for 5 minutes to pellet the debris before transferring 8 µl of the clear supernate. 2. Mix content by gently shaking on a vortex machine at low speed for 5-10 seconds (i.e. shake speed of 1-2 on a Vortex 2 Genie equipped with 96-well plate adapter). Pµlse spin and incubate for 5 minutes at 37 C. Incubate an additional 15 minutes at 72 C. Note: The two temperatures of this incubation step can be programmed into PCR thermocyclers. Do not use the heated lid option if available. 3. After the 72 C incubation, the PCR products are clean and ready for sequencing. -If sequencing is done on the same day store samples at 4 C until used. Follow sequencing guidelines below. -If sequencing will be at a later date stabilize the templates by drying in a vacuum centrifuge at room temperature. Before sequencing add 10 µl ddh 2 O and spin for 10 minutes at room temperature. Follow the sequencing guidelines below. Any Questions? Call Technical Support at (800)

9 Note: the presence of inert stabilizers, which are activated upon drying, ensures optimal sequencing results for up to two weeks if the cleaned/stabilized PCR templates are stored at room temperature and several month if stored at 4 C or -20 C. Guidelines for sequencing reactions Amount of PCR products to use per sequencing reaction for the various sequencing machines. Use the same quantity for full and 1 /2 reactions. PE Applied Biosystems Beckman ABI 373 ABI 377 ABI 3700 CEQ µl 1-2 µl 1-2 µl 1-2 µl 4. Troubleshooting 1. Electropherogram with top heavy signal: dilute the cleaned PCR template 2-5 fold in H 2 O prior to sequencing. 10 visit us on the web at

10 Contact Us: In North America 2251 Rutherford Rd. Carlsbad, CA USA Tel.: (800) or (760) Fax: (760) Internet: In Europe Parc d Innovation BP 72, Illkirch Cedex-France Tel.: +33 (0) Fax: +33 (0) Internet: techservfr@qbiogene.com