Notes: These authors describe map-based cloning tools for Chlamydomonas reinhardtii, (green yeast). In this case-based study of existing and projected resources for C. reinhardtii mapping, mcd4 and mcd5 mutants were mapped by crossing to the infertile strain known as Chlamydomonas grossii, S1-D2, with several known marker sites. To reduce the time and expense of mapping, bulked segregant analysis (BSA) and marker duplexing were evaluated. In BSA, DNAs from multiple segregating progeny (up to six in this study) are combined, and results from PCR-based markers are examined for significant bias from a roughly equal contribution from each parent. This case study used 57–72 markers to span the 1,107-cM genome using approximately 3,350 PCR amplifications. The researchers picked single colonies from a plate and placed each colony in a hypotonic EDTA solution. After boiling for 5 minutes, the supernatant was collected by centrifugation and the DNA quantitated. Twenty nanograms of DNA thus isolated was used as the template for PCR. The reaction included GoTaq^® DNA polymerase with the enhancers 8.5%glycerol and 0.83% formamide in a 30µl reaction. Forty cycles of amplification were performed and the results were analyzed by agarose gel electrophoresis. The various amplification reactions included the use of BSA and both monoplex and duplex reactions with amplicons of 90bp–625bp. (3279)

Notes: The presence or absence of intercellular adhesion genes (icaA and icaD) in various strains of Staphylococcus epidermidis isolated in medical facilities was investigated using PCR amplification and GoTaq^® DNA Polymerase. Each reaction contained 150ng of template. GoTaq^® Green Reaction Buffer was used in the PCR. (3359)

Notes: GoTaq^® DNA Polymerase was used to amplify various portions of the rDNA isolated from microsporidia-infected brysozoans. The sequence of these amplimers was used in the construction of a phylogenetic tree. (3379)

Notes: In vitro cultures of P. alba were incubated in growth chambers under various conditions. The axenic plants of P. alba were sprayed with either methyl jasmonate (100 lM), methyl salicylate (1mM), or ethanol as a control, and then grown under illumination at 18 hours/day for 24 hours at 25°C. Total RNA was subjected to semi-quantitative RT-PCR using GoTaq^® DNA polymerase and primers for hybrid poplar isoprene synthase. For normalization, actin was used as an external standard. (3355)

Notes: To examine the role of Rac3 in nervous system development, knockout mice were created. Total RNA from postnatal day 0 (P0) and day 9 (P9) brains from wild-type (+/+), heterozygous (+/-), and knockout (-/-) mice was reverse transcribed and a ~260bp product amplified by PCR using GoTaq^® DNA Polymerase and primers for either Rac3 or Rac1 (a Rac3 homolog). No amplification product for Rac3 was observed in the knockout mice. (3328)

Notes: GoTaq^® DNA Polymerase was used in an RT-PCR. Aliquots of RNA were transcribed into cDNA and amplified with GoTaq^® DNA Polymerase using primers specific for Rac1. (3351)

Notes: GoTaq^® DNA Polymerase was used in PCR genotyping of Giardia intestinalis. Primers were designed around a 177 bp sequence of the glutamete dehydrogenase gene (gdh). Typing was based on previously reported assemblages of gdh from cats, dogs, cows and monkeys. PCR was performed in a total reaction volume of 25μl using 1.25 units of GoTaq^® DNA Polymerase.
(3364)

Notes: Researchers tested several snail samples from various species for the presence of microsporidia infection. PCR tests were performed with GoTaq^® DNA Polymerase and primers specific to microsporidia 16S DNA sequences. Isolated snail/microsporidia DNA was amplified in 25µl reactions with 0.625 units of GoTaq^® DNA Polymerase. (3378)

Notes: M-MLV Reverse Transcriptase was used to make cDNA from differentiated THP-1 cell total RNA. For these reverse transcription experiments the authors used 5 µg of total RNA, oligo dT and RNasin^®. GoTaq^® DNA Polymerase was used to amplify the cDNA from the reverse transcription reactions. PCR products were separated by gel electrophoresis and analyzed by ethidium bromide staining and band densitometry. (3357)

Notes: GoTaq^® DNA Polymerase was used in a RT-PCR. The 50μl PCR reactions contained 0.25μl cDNA, 12.5pmol primer, 0.025mM dNTPs, 2.5mM MgCl₂ and 0.25μl of GoTaq^® DNA Polymerase (5U/μl). PCR was performed using the following conditions: 94°C for 3 minutes followed by 20 cycles (94°C for 30 seconds, 55°C for 30 seconds and 72°C for 30 seconds), followed by 72°C for 7 minutes and held at 4°C. Products were visualized on an agarose gel. (3352)

Notes: To examine the effect of the exoribonuclease Rat1p on processing of ribosomal RNAs (rRNA), total RNA was isolated from several yeast strains with altered RAT1 expression. One microgram of DNase-treated total RNA was reverse transcribed in a 20µl reaction. Subsequently, 0.25µl of cDNA product was amplified using 0.25µl of GoTaq^® DNA polymerase in a total volume of 50µl. The RT-PCR products were separated on 2% agarose gels and stained using ethidium bromide. (3330)

Notes: To test the activity of torI as a prophage excisionase, the chloramphenical aceytltransferase gene was introduced into a non-coding region of the prophage KplE1 in a strain of E. coli containing a torI-encoding plasmid. Expression of torI was induced by IPTG and the cells plated on either chloramphenical or ampicillin. GoTaq^® DNA polymerase was used in a PCR test to confirm prophage DNA excision from randomly chosen ampicillin-resistant colonies. (3329)

Notes: GoTaq^® Polymerase was used in this study investigating the role of TorI (Tor inhibition protein) in prophage DNA excision. PCR products were amplified from E. coli strains carrying a plasmid-encoded torI gene, and were analyzed by gel electrophoresis. (3350)

Notes: To examine why there are two splicing systems present in multicellular organisms, the authors used morpholino oligomers complementary to the branch-site recognition elements of U2 or U12 small nuclear RNA to specifically suppress the splicing function in EL4 and Jurkat cells. After transfection with the morpholino oligos, the cells were harvested, and total RNA was isolated followed by DNase-treatment. In a 20µl reverse transcription reaction, 0.8µg total RNA was reverse transcribed with 100ng oligo(dT)₁₅ primer and 200 units of M-MLV Reverse Transcriptase, RNase H Minus. PCR was then performed using 5µl of the diluted (1:10) reverse transcriptase reaction with 2.5 units GoTaq^® DNA polymerase in a 50µl reaction for 28–31 cycles. (3278)

Notes: PCR was performed in 50μl GoTaq^® Reaction Buffer containing 5μl of diluted (1:10) reverse transcriptase reactions, 250mM each dNTP, 10 pmol primers and 2.5 U GoTaq^® DNA Polymerase. A total of 28 cycles were performed for amplification of P120 minigene transcripts, and 31 cycles were performed for endogenous P120 and ADPRT transcripts. Each cycle consisted of 95°C for 1 min, 59°C (P120) or 58°C (ADPRT) for 1 min and 72°C for 1.5 min. (3353)

Notes: GoTaq^® DNA Polymerase was used to amplify cDNAs created from total RNA of various yeast strains. Primers specific to ACT1 mRNA and 27S rRNA were used to analyze transcription levels under various conditions.  (3226)

Notes: GoTaq^® DNA Polymerase was used in PCR amplifications to genotype zfarnt2 -/- zebrafish mutants with a specific set of primers. The zfarnt2 gene encodes a receptor that is important for mediating chemical toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in zebrafish. (3192)

Notes: In this study, GoTaq^® DNA Polymerase was used to PCR amplify a region of the Disrupted-in-Schizophrenia-1 (DISC1) gene from patient samples. Specific primers were used in the amplification reactions to test for polymorphisms in the region. (3128)

Notes: Researchers used GoTaq^® DNA polymerase to amplify 139bp and 815bp regions of hOST-PTP cDNA for detection and probe synthesis. The full-length 4006bp cDNA was amplified with Pfu DNA Polymerase. Fragments were gel purified using the Wizard^® SV Gel and PCR Clean-Up System prior to cloning into the pGEM^®-T Easy Vector.  The Prime-a-Gene^® Labeling System was used to make ³²P-dCTP labeled probes, which were used to screen cDNA clones. (3111)

Notes: MCF-7 cells were incubated for 24 hours in PRF-SFM and then detached and plated on uncoated dishes or dishes coated with 2μg/cm² poly-L-Lysine (P-Lys) in PBS, 30μg/ml fibronectin (Fn) in PBS or 30μg/ml type IV Collagen (Col) in 10mM acetic acid. The cells plated on uncoated dishes were treated both with and without 10nM estradiol (E2). After 24 hours, total cellular RNA was extracted and reverse transcribed using M-MLV reverse transcriptase. Briefly, reverse transcription was performed on 1μg of total RNA in a final volume of 10μl by incubation at 37°C for 30 min with 200U of M-MLV reverse transcriptase, 0.4μg oligo-dT, 0.5μM dNTP and 24U RNasin^® Ribonuclease Inhibitor, followed by heat denaturation for 5 minutes at 95°C. Subsequent PCR analysis was performed on 1μl of the RT product in a final volume of 25μl. Primers were used to amplify the 210bp fragment of PS2 and the 304 bp fragment of cathepsin D. Amplification of 408bp of ribosomal RNA 36B4 was performed as control. The PCR mixture consisted of 1.25U GoTaq^® DNA Polymerase, 1X PCR buffer (10mM Tris-HCl, 50mM KCl), 2.5mM MgCl₂, 0.2mM each dNTP, 0.6μM of each PS2 primer or cathepsin D primer and 0.2μM of each 36B4 primer. PCR was performed for 20 cycles at 95°C for 1 minute, 59°C for 2 minutes and 72°C for 1 minute. Ten microliters of the PCR products were separated on a 1.2% agarose gel. (3377)

Notes: GoTaq^® DNA Polymerase was used to amplify a portion of Human Papillomavirus 16 (HPV 16) from cervical carcinoma-derived genomic DNA. The amplified region of HPV 16 was then purified and used in sequencing reactions. (3358)

Notes: GoTaq^® DNA Polymerase was used to amplify Green Fluorescent Protein and β-actin cDNA. This paper describes use of a “Touchdown” PCR procedure for amplification of the β-actin cDNA. Gels are presented displaying the amplification products from the reporter constructs and cells used in the experiments. (3129)

Notes: COS-7 cells were harvested and total RNA isolated 24 hours after transfection with plasmids expressing trans-activating proteins. Five micrograms of RNA was reverse transcribed with oligo(dT) primer using the M-MLV Reverse transcriptase. One tenth of the reaction volume (2μl) was then used as template in PCR to amplify a gene presumed to be controlled by the trans-activators (pS2; 210bp) or a control gene (36B4 ribosomal phosphoprotein; 408bp). The PCR products were amplified using GoTaq^® DNA polymerase. (3203)

Notes: Total RNA was isolated from leaf tissue from Arabidopsis plants treated with rhizobacteria Serattia marcescens strain 90-166, 300μM benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH), 100μM jasmonic acid (JA), or water for 2 days. Reverse transcription reactions were performed on 1–5μg of total RNA using 200 units of reverse transcriptase, 500ng oligo d(T)_12–16mer primer and 500μM dNTPs in a final volume of 20μl. Semi-quantitative RT-PCR was performed in a final volume of 75μl using 1.5μl of cDNA, 1X PCR buffer (with 1.5 mM MgCl₂), 200μM dNTP, 200nM of each gene specific primer (primers were designed from the Arabidopsis PR-1 gene) and 1.5 units of GoTaq^® DNA Polymerase. To ensure that only host genes and not the viral RNA transcripts were amplified, the RT reactions were performed using oligo d(T) primers. As a loading control, parallel reactions using elongation factor primers were performed. PCR conditions for all genes were as follows: One cycle of 4 minutes at 94°C, 30 seconds at 55°C and 30 seconds at 72°C was followed by 34 cycles of 30 seconds at 92°C, 30 seconds at 59°C and 30 seconds at 72°C. A 5μl aliquot was removed from each reaction after 25, 30, and 35 cycles. These aliquots were analyzed on a 1% agarose gel stained with ethidium bromide. (3371)

Notes: GoTaq^® DNA Polymerase was used in a multiplexed genotyping reaction with three primers to differentiate wild-type, heterozygous, and mutant transgenic mouse alleles simultaneously. The wild-type target was 670bp and the mutant or knockout target was 1,030bp. Various other amplimers (1.4kb, 471bp, & 311bp) were subcloned with the aid of the pGEM^®-T Easy Vector System. (3360)