Partially dissecting the amino acid sequence of the peptide by about 20 residues, the PCR reaction with PCR primers encoding the ends of the peptide enables the length of the PCR product to be detected. The cDNA obtained by this method is only a few tens of base pairs, but it will be an important breakthrough for subsequent screening of genes. Source: Handbook of Protein Technology
Operation method
basic program
Materials and Instruments
PK-120 Move See "Other" for "Reagents" required for the experiment. 1. Primer design The primer design is based on the longest sequence K-15,16 in the amino acid sequence of PK-120 partially disassembled peptide: 1.1 Considering the combination of all codes. 1.2 the frequency of use of the human code, and the design of PCR primers for the positive and negative strands such that 50 bp of the PCR product is the target cDNA. 2. 2. PCR amplification Poly(A) + poly(A) from liver source was amplified. poly(A)+ RNA from the liver. RNA from the liver, a 0.1ug poly(A)+ cDNA was synthesized using the Gene Amp RNA PCR kit as described below. + RNA RNA in 5 mmol/L MgCl2: 1× PCR buffer II. 2 mmol/L dNTPs. 1u of RNase inhibitor. 2.5 umol/L random hexamers 2.5 u reverse transcriptase. The total volume was 20 ul. The reaction was performed on a PCR amplifier with the following PCR parameters: 25℃ for 10 min, 42℃ for 30 min, and 42℃ for 30 min, 99℃ for 5 min, and PCR parameters were: 25℃ for 10 min, 42℃ for 30 min, 99℃ for 5 min, and 25℃ for 5 min. The cDNA was synthesized in the first cycle of the reaction. Add 2 mol/L MgCl2, 1×PCR buffer Ⅱ and 1 nmol of 3' primer and 5' primer to the reaction solution. The total volume was 100 ul. Add 1 nmol of 3' primer and 5' primer to the mix, total volume is 100 ul. 94℃ for 1 min. 37℃～42℃, 2 min. 72℃, 2 min. 40 cycles. 72℃, 7 min, 1 cycle. 3. Gel electrophoresis and DNA extraction After purification of the PCR products, a 10% polyacrylamide gel electrophoresis was performed in 0.25×TBE, and the molecular weight standard was the plasmid pBR322 digested with the restriction enzyme Msp1. At the end of the electrophoresis, the gel was stained with ethidium bromide, and a 50-bp PCR product was visualized by UV irradiation. The 50-bp band was cut off and extracted from the gel. The 50-bp band was excised and the supernatant was recovered by centrifugation at 1200 r/min for 10 min at 4 ℃ in extraction buffer at room temperature overnight. The procedure was repeated twice, precipitated twice with ethanol, solubilized in TE solution, loaded onto a Sephadex-50 column, and then eluted with ethanol precipitation of the fractions, which were suspended in TE solution. 4. 4. Determination of base sequences The PCR products were extracted and subcloned into PCR II vector for base sequence determination. The sequence of the 50 bp PCR product amplified from liver poly mRNA using primers S15-i and A15-i as template was identical to the base sequence predicted from the amino acid sequence. This indicated that the cDNA encoded the parent partially disassembled polypeptide. Common Problems Reagents: Sephadex-50 Gene Amp RNA PCR Kit Synthesizing Oligonucleotides with BIO-SYNTHESIS, INC. Extraction solution 750 mmol/L ammonium acetate-10 mmol/L magnesium acetate-0.1 mmol/L EDTA-0.1% SDS-1% TE Saturated Phenol 25ug/ml yeast tRNA Other reagents are commercially available For more product details, please visit Aladdin Scientific website.
Sephadex-50 Gene Amp RNA PCR Kit Synthetic Oligonucleotide Extract Yeast tRNA
PCR Amplifier