The DNA fragments are amplified in a PCR step by using the following two linker-specific primers: Primer1: 5'-biotin-CTATAGAAGAGTCCTGACCTAGG-3'; Primer2: 5'-biotin- CGGTCCTAAGGTAGCGACCTAG-3'. [number_of_1stPCR_tubes] parallel PCRs are performed in a total volume of 50µl. After incubation at 94 degrees Celsius for 1min, [number_of_1stPCR_cycles] cycles PCR are performed for 30sec at 94 degrees Celsius, 20sec at 55 degrees Celsius, 20sec at 70 degrees Celsius, followed by 5min at 72 degrees Celsius. The resulting PCR products were pooled, purified, isopropanol precipitated, and finally resuspended in 0.1xTE buffer.<br /><br />The PCR products are purified on a 12% polyacrylamide gel. The appropriate 75bp band is cut out of the gel, crushed, and added with an elution buffer for over night at room temperature. The extracted tags are filtrated with MicroSpin? Columns. We added an elution buffer to the gel, rotated the tube at room temperature for 30min, repeated this extraction step more 3 times. The extracted tags are precipitated with ethanol and dissolved in 30µl of 0.1xTE buffer. The concentration was measured with Picogreen.<br /><br />Purified bands are PCR-amplified once more in a total of 100 µl by using [amount_of_cDNA_tags] ng of cDNA-tags. [number_of_2ndPCR_tubes] tubes were heated to 94 degrees Celsius for 1min, then [number_of_2ndPCR_cycles] cycles were performed for 30 sec for 94 degrees Celsius, 20 sec for 55 degrees Celsius, 20 sec for 70 degrees Celsius, followed by a final elongation at 72 degrees Celsius for 5min. The PCR products are pooled , purified, ethanol precipitated and finally redissolved in 0.1xTE buffer. To eliminate excess primers, the PCR products are further purified with MinElute? columns(QIAGEN), ethanol precipitated and finally redissolved in 0.1x TE buffer. The concentration is measured with Picogreen. The [amount_of_tags_for_XmaJI_cut] of purified PCR products are digested with XmaJI in some tubes (2µg/tube), followed by proteinaseK treatment. The volume of one tube was 200µl. The desired CAGE tags are separated from the free DNA ends cut off during restriction by incubation with streptavidin-coated magnetic beads, which retain the biotin-labeled DNA ends. After purification (phenol/chloroform extraction and ethanol precipitation and dissolved in 45µl of TE.<br /><br />The tags are further purified on a 12% polyacrylamide gel. The appropriate 37bp band is cut out of the gel, crushed, and eluted with the previously used elution buffer for O/N at room temperature, followed by ethanol precipitation. The tags were dissolved in 6µl of 0.1xTE buffer. The concentration was measured with picogreen.<br /><br />The [amount_of_ligated_CAGE_tags] ng of CAGE tags were ligated to form concatemers by adding to tags and 1/20 amount of tags of 454 adaptors A/B as described in the original publication (Nature. 2005 Sep 15;437(7057):376-80). The sample was purified with GFX column to eliminate short concatemers. Then the library was sequenced with Genome Sequencer FLX System.
<HTML><HEAD><TITLE>454CAGE_step2</TITLE><meta http-equiv="content-type" content="text/html; charset=UTF-8"/><script type="text/javascript" src="/sw/js/panel/panelDescription.js"></script></HEAD><BODY>"The DNA fragments are amplified in a PCR step by using the following two linker-specific primers: Primer1: 5'-biotin-CTATAGAAGAGTCCTGACCTAGG-3'; Primer2: 5'-biotin- CGGTCCTAAGGTAGCGACCTAG-3'. [number_of_1stPCR_tubes] parallel PCRs are performed in a total volume of 50µl. After incubation at 94 degrees Celsius for 1min, [number_of_1stPCR_cycles] cycles PCR are performed for 30sec at 94 degrees Celsius, 20sec at 55 degrees Celsius, 20sec at 70 degrees Celsius, followed by 5min at 72 degrees Celsius. The resulting PCR products were pooled, purified, isopropanol precipitated, and finally resuspended in 0.1xTE buffer.<br /><br />The PCR products are purified on a 12% polyacrylamide gel. The appropriate 75bp band is cut out of the gel, crushed, and added with an elution buffer for over night at room temperature. The extracted tags are filtrated with MicroSpin? Columns. We added an elution buffer to the gel, rotated the tube at room temperature for 30min, repeated this extraction step more 3 times. The extracted tags are precipitated with ethanol and dissolved in 30µl of 0.1xTE buffer. The concentration was measured with Picogreen.<br /><br />Purified bands are PCR-amplified once more in a total of 100 µl by using [amount_of_cDNA_tags] ng of cDNA-tags. [number_of_2ndPCR_tubes] tubes were heated to 94 degrees Celsius for 1min, then [number_of_2ndPCR_cycles] cycles were performed for 30 sec for 94 degrees Celsius, 20 sec for 55 degrees Celsius, 20 sec for 70 degrees Celsius, followed by a final elongation at 72 degrees Celsius for 5min. The PCR products are pooled , purified, ethanol precipitated and finally redissolved in 0.1xTE buffer. To eliminate excess primers, the PCR products are further purified with MinElute? columns(QIAGEN), ethanol precipitated and finally redissolved in 0.1x TE buffer. The concentration is measured with Picogreen. The [amount_of_tags_for_XmaJI_cut] of purified PCR products are digested with XmaJI in some tubes (2µg/tube), followed by proteinaseK treatment. The volume of one tube was 200µl. The desired CAGE tags are separated from the free DNA ends cut off during restriction by incubation with streptavidin-coated magnetic beads, which retain the biotin-labeled DNA ends. After purification (phenol/chloroform extraction and ethanol precipitation and dissolved in 45µl of TE.<br /><br />The tags are further purified on a 12% polyacrylamide gel. The appropriate 37bp band is cut out of the gel, crushed, and eluted with the previously used elution buffer for O/N at room temperature, followed by ethanol precipitation. The tags were dissolved in 6µl of 0.1xTE buffer. The concentration was measured with picogreen.<br /><br />The [amount_of_ligated_CAGE_tags] ng of CAGE tags were ligated to form concatemers by adding to tags and 1/20 amount of tags of 454 adaptors A/B as described in the original publication (Nature. 2005 Sep 15;437(7057):376-80). The sample was purified with GFX column to eliminate short concatemers. Then the library was sequenced with Genome Sequencer FLX System."</BODY></HTML>
454CAGE_step2