선택한 단어 수는 입니다.
최소 단어 이상 선택하여야 합니다.
최소 단어 이상 선택하여야 합니다.
선택한 단어 수는 30입니다.
최대 10 단어까지만 선택 가능합니다.
최대 10 단어까지만 선택 가능합니다.
제안 방법
- These DNA probes were synthesized and labeled with [32P] dCTP as described by the procedures of the Prime-a-Gene® Labeling System (Promega, Madison, Wl). Hybridization was performed with the labeled probes for 14 h at 42℃ in hybridization buffer (50% formamide, 1.0 M NaCI, 10% (w/v) dextran sulfate, 1% SDS and 0.1 mg/mL denatured salmon sperm DNA). The membrane was wa아led twice in 2x SSC, 0.
- The first strand cDNA was generated from 1 gg of the total RNA using a RNA PCR kit (lakara, Kyoto, Japan), and it was then amplified. The PCR amplications were performed in a total 25 μL volume containing 5 |iL cDNA, 1 mM dNTP, 2.5 mM MgCI2, 12.5 pm이 of primers and 0.6 1) faq DNA polymerase for 30 cycles under the following conditions: denaturation at 94℃ for 30 s, annealing at 57-63℃ for 30 s, and extention at 72℃ for 30 s. The PCR samples were electrophoresed on 1.
- Differential display (dd) PCR was performed using the Delta Differential Display system (Clontech, Palo Alto, CA) according to the manufacturer's instructions in the presence of [ot35S] dATP. The amplified radioactive products were resolved by electrophoresis on a denaturing urea 5% polyacrylamide gel, After overnight autoradiography, the differentially expressed bands were identified by a comparison of the products that were obtained when using the RNA from BMMCs and CTMCs. The products fo나nd to be differentially expressed in a reproducible fashion were reamplified and subcloned into pCR2.
- The amplified radioactive products were resolved by electrophoresis on a denaturing urea 5% polyacrylamide gel, After overnight autoradiography, the differentially expressed bands were identified by a comparison of the products that were obtained when using the RNA from BMMCs and CTMCs. The products fo나nd to be differentially expressed in a reproducible fashion were reamplified and subcloned into pCR2.1-TOPO vectors using the TA cloning system (Invitrogen). The plasmids containing the amplified inserts were p니rified, the sequences of the insert were determined by automated dideoxy sequencing at Yeungnam University and they were compared to known sequences using the BLAST n아work of the National Center for Biotechnology Information.
대상 데이터
- The expression levels of each clone were also examined by RT-PCR using the specific primers that were derived from the ruicleotide sequences of each clone. The confirmed unknown genes were #8 (Accession number NM_172450; hypothetical protein), #17 (Accession number: BC019462), and #35 (Accession number: AK089766; hypothetical protein). Tb date, we do not know the functions of those unknown genes induced in the CTMCs, and we are trying to isolate the full length of cDNA for further investigation.
이론/모형
- The total RNA was prepared from BMMCs and CTMCs using Trizol (Invitrogen). Differential display (dd) PCR was performed using the Delta Differential Display system (Clontech, Palo Alto, CA) according to the manufacturer's instructions in the presence of [ot35S] dATP. The amplified radioactive products were resolved by electrophoresis on a denaturing urea 5% polyacrylamide gel, After overnight autoradiography, the differentially expressed bands were identified by a comparison of the products that were obtained when using the RNA from BMMCs and CTMCs.
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