Analyses of the samples were carried out according to techniques described by Surh et al.31 (link) PCR was performed in a final volume of 30 µL using the Perkin Elmer GeneAmp PCR system. The forward, 103, 5′—CCA GTT CAC AAA CCG CTC CGA GCG TG—3′ and reverse, 96, 5′—GGT GCG TGG AGG ATG GAA CAC GGA C—3′ primers were used. The PCR conditions were set as follows: initial denaturation at 96°C for 5 min, followed by 25 cycles of denaturation, annealing and extension at 96°C, 62°C and 72°C, respectively, for a period of 1 min for each step. Final extension was performed at 72°C for 7 min. The PCR products were sized by gel electrophoresis on 1.5% agarose gel, at 100 V for 45 min. The separated products were cut out from the gel, purified using the QIAquick gel extraction kit (QIAGEN, Hilden, Germany) and sent to a service laboratory for sequencing to determine the exact number of CTG repeats.
Geneamp pcr system
Manufactured by PerkinElmer
Sourced in Italy, United States
The GeneAmp PCR System is a thermal cycler used for DNA amplification by the Polymerase Chain Reaction (PCR) technique. It precisely controls the temperature and timing of the thermal cycling process, which is essential for the exponential replication of DNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
Pcr buffer, by Thermo Fisher Scientific (2 mentions)
Universal primer oligo dt 15, by Promega (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
Dna free dnase, by Thermo Fisher Scientific (1 mentions)
Iq sybr green master mix, by Bio-Rad (1 mentions)
Abi prism 7300, by Thermo Fisher Scientific (1 mentions)
Amplitaq gold polymerase, by PerkinElmer (1 mentions)
Superscript reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
6 protocols using geneamp pcr system
1
Determination of CTG Repeat Number
2
Quantitative RT-PCR for CXCL12 Expression
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Total RNA was prepared from cells using the TRIzol reagent (Gibco), according to the manufacturer's instructions. After purification, 1-µg amounts of RNA were reverse-transcribed using SuperScript reverse transcriptase (Gibco) and the universal primer oligo (dT)15 (Promega, Madison, WI). In each reaction, 1 µL of cDNA was added to 24 µL of PCR buffer (Gibco) supplemented with 2 mM MgCl2, 0.2 µM of each primer, and 1 U Koma Taq polymerase (Koma International, Seoul, Korea). Using a GeneAmp PCR system (Perkin Elmer, Norwalk, CT), 30 cycles of 1 min at 94℃, 45 sec at 55-65℃, and 1 min at 72℃, were performed. The following primers were used: human CXCL12 (sense, AGA ATT CAT GAA CGC CAA GG; anti-sense, AGG ATC CTC ACA TCT TGA ACC); and human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (sense, CAT GTG GGC CAT GAG GTC CAC CAC; antisense, TGA AGG TCG GAG TCA ACG GAT TTG GTC).
3
PCR Temperature Cycling Program
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The temperature cycling program used with a Perkin-Elmer Gene Amp PCR system (model 2400) was as follows: one cycle at 94 °C for 5 min followed by 30 cycles consisting of one step of denaturation (94 °C) for 40 sec, one step of annealing (37 °C) for 1 min, followed by one step of synthesis (72 °C) for 2 min and a final extension step 72 °C for 7 min and finally 4 °C infinitive (Williams et al., 1990 (link)).
4
RNA Extraction and RT-PCR Analysis of MR and GR
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Total RNA extraction from AHP cells and reverse transcription to cDNA from 3 μg RNA were performed as previously described (39 (link)). Nine microliters of cDNA were amplified by standard polymerase chain reaction (PCR) in a 50 μl volume using AmpliTaq Gold Polymerase in a GeneAmp PCR System (Perkin Elmer, Milano, Italy) as described (10 (link)). The following primer pairs were used for both RT-PCR and real time PCR: MR (40 (link)), forward 5′-TACGACAATTCCAAGCCCGACACC-3′, reverse 5′-TACCTTGGCCCACTTCACGACCTG-3′ (99 bp) (NM_013131). GR (40 (link)), forward 5′-AGGGGAGGGGGAGCGTAATGG-3, reverse 5′-CCTCTGCTGCTTGGAATCTGC-3′ (119 bp) (AY293740); rat 18S rRNA, forward 5′-GTGGAGCGATTTGTCTGGTT-3′, and reverse 5′-CGCTGAGCCAGTTCAGTGTA-3′(X01117). Amplification for 18S rRNA subunit was used as internal control. For real-time PCR, cDNA was treated with DNA-free DNAse (LifeTech, Monza, Italy). Real-time PCR was performed with 50 ng cDNA, 100 nmol/L of each primer and the IQ-SYBR-green mastermix (Bio-Rad, Milano, Italy) using the ABI-Prism 7300 (Applied Biosystems).
5
Quantitative RT-PCR Analysis of Chemokine and Receptor Expression
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Total RNA was prepared from cells using TRIzol reagent (Gibco-BRL Life Technologies, Grand Island, NY, USA), according to the manufacturer's instructions. After purification, 1 µg RNA was reverse-transcribed using SuperScript reverse transcriptase (Gibco-BRL Life Technologies, Grand Island, NY, USA) and the universal primer oligo (dT)15 (Promega, Madison, WI, USA). In each reaction, 1 µL of cDNA was added to 24 µL PCR buffer (Gibco-BRL Life Technologies, Grand Island, NY, USA) supplemented with 2 mM MgCl2, 0.2 µM of each primer, and 1-U Koma Taq polymerase (Koma International, Seoul, Korea). Using a GeneAmp PCR system (Perkin Elmer, Norwalk, CT, USA), 30 cycles of 1 min at 94℃, 45 sec at 55-65℃, and 1 min at 72℃ were performed. The following primers were used: human CXCL12 (sense, AGA ATT CAT GAA CGC CAA GG; anti-sense, AGG ATC CTC ACA TCT TGA ACC); human CXCR4 (sense, AAT CTT CCT GCC CAC CAT CTA CTC C; antisense, GCG GTC ACA GAT ATA TCT GTC ATC TGC C); human CXCR7 (sense, ACG TGG TGG TCT TCC TTG TC; antisense, AAG GCC TTC ATC AGC TCG TA); and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH; sense, CAT GTG GGC CAT GAG GTC CAC CAC; antisense, TGA AGG TCG GAG TCA ACG GAT TTG GTC).
6
Cloning of CfrBI R-M System Genes
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The plasmid pBGM5, carrying the total nucleotide sequence of the CfrBI R-M system, was used as a template in polymerase chain reaction (PCR) to clone cfrBIM and gene encoding CfrBI restriction endonuclease (cfrBIR).
The “reverse” 29-mer primer (P1, see Section2.3 ) used to clone the cfrBIM gene contains a BamHI site (underlined) immediately after the ATG start codon; the “forward” 30-mer primer (P2, see Section 2.3 ) contains a PstI site (underlined) just after the termination codon (TAA) of cfrBIM. The cfrBIM gene was amplified as an ATG-lacking 1127-bp fragment. This PCR fragment was cloned into a BamHI-PstI-digested pQE 30 expression vector. The plasmid with a functionally active cfrBIM gene was named pMet17.
The “forward” 35-mer primer (P3, see Section2.3 ) used to clone cfrBIR contains a BamHI site (underlined) just after the ATG start codon; the “reverse” 31-mer primer (P4, see Section 2.3 ) contains a HindIII site (underlined) just after the termination codon (TAA) of cfrBIR. The cfrBIR gene was amplified as a 1064-bp ATG-lacking fragment. This PCR fragment was cloned into a BamHI-HindIII-digested pQE 30 expression vector. The plasmid with a functionally active cfrBIR gene was named pRes10.
All PCR experiments were performed using a Gene Amp PCR System (Perkin Elmer). The PCR operating parameters were: 30 cycles (94 °C for 30 s, 55 °C for 30 s and 72 °C for 60 s).
The “reverse” 29-mer primer (P1, see Section
The “forward” 35-mer primer (P3, see Section
All PCR experiments were performed using a Gene Amp PCR System (Perkin Elmer). The PCR operating parameters were: 30 cycles (94 °C for 30 s, 55 °C for 30 s and 72 °C for 60 s).
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