Notes: To demonstrate an interaction of activated CaM KII with the postsynaptic density protein densin-180, a gst-fusion protein of densin was produced and reacted with autophosphorylated CaM KII. The glutathione matrix-bound material was eluted and immunoblotted for phospho-Thr286-CaM KII. The autophosphorylated CaM KII was pulled down on the Glutathione only when the densin-GST fusion was present. The phosphoCaM KII was detected with the Anti-ACTIVE^® CaM KII pAb. The clone of densin was generated by RT-PCR of rat forebrain total RNA with the Access RT-PCR System. (0076)

Notes: Several cancer-associated proteins, such as the tumor suppressor p53 and the oncoproteins c-ABL and hdm2, have been shown to shuttle between the nucleus and the cytoplasm. Due to this information and the controversy regarding BRCA1 localization, the authors wanted to determine whether BRCA1 is also capable of nuclear-cytoplasmic shuttling.

To this end, a plasmid encoding untagged BRCA1 was created by subcloning the full-length BRCA1 cDNA into a vector as a NotI/ClaI fragment. To generate pYFP-BRCA1, a DNA fragment encoding the yellow fluorescent protein (YFP) was amplified by polymerase chain reaction (PCR) and inserted in frame at the end of the BRCA1 cDNA in the plasmid, using the unique NotI restriction site.

Human breast cancer cell lines T47D and MCF-7, the human breast epithelial cell line HBL-100, and mouse NIH3T3 fibroblasts were seeded onto glass coverslips and transfected at 50–70% confluency with 0.5–2µg of plasmid DNA using the FuGENE® 6 Transfection Reagent. After 48 hours, cells were fixed and processed for fluorescence microscopy. (4283)

Notes: The pTARGET™ Mammalian Expression Vector was used to clone and express MIG (monokine-induced by Interferon-gamma) and IP-10 (interferon-inducible protein 10) in A549 human adenocarcinoma cells. Stable transfectants were obtained through G418 selection. (2058)

Notes: Poly(A)⁺ RNA was purified from total RNA by the PolyATtract^® mRNA Isolation System. PCR products were purified by agarose gel electrophoresis and were cloned into pGEM^®-T and pGEM^®-T Easy Vector. Genes were obtained by RT-PCR using the Access RT-PCR System. (1097)

Notes: The Access RT-PCR System was used to amplify Vα₁₀Jα₂₄ mRNA of the TCR alpha chain and the resulting product was cloned with the pGEM^®-T Vector System. (0734)

Notes: Taq DNA Polymerase was used extensively to clone the BLINaC cDNA. The resulting clone was put into the pCI Mammalian Expression Vector. Total RNA was isolated from rat liver and primary hepatocytes with the SV Total RNA Isolation System. The RNA was used for RT-PCR. Northern blotting was performed with poly-A(+) RNA isolated with the PolyATtract^® mRNA Isolation System. (0472)

Notes: The pSP73 Vector was used for routine subcloning. The pGEM®-T Vector System was used for subcloning of PCR products. (1320)

Notes: Subunits of the cDNA for methionine adenosyltransferase were amplified from a plasmid using Taq DNA Polymerase and directly cloned into the pTARGET™ Mammalian Expression T-Vector. The sequence of the cloned inserts were confirmed with the fmol^® DNA Cycle Sequencing System. The constructs were transiently transfected into COS-1 cells with the TransFast™ Reagent at a 1:1 ratio. Excellent detail is provided for the transfection. (1094)

Notes: The complete cDNA for the CD1.1 gene was amplified and subcloned into the pTARGET™ Mammalian Expression Vector. The 1020bp, 339 amino acid protein was stably expressed in CHO cells following selection with G-418 sulfate. Expression was confirmed by Northern blot and FACS analysis with an mAb to the CD1.1 protein. The pGEM®-T Vector System was used for routine cloning of both PCR and RT-PCR products. The Prime-a-gene® Labeling System was used create probes for cosmid library screening. (1303)

Notes: The pGEM^®-3Zf(+) Vector was used for routine subcloning and the resulting plasmid was used as a template for PCR. (0114)

Notes: Total RNA was isolated from 3-day-old mung bean hypocotyls using the SV Total RNA Isolation System. RT for RT-PCR was performed with the Reverse Transcription System. (0825)

Notes: Before RT-PCR reactions, the RNA template was treated with RQ1 RNase-free DNase. (0776)

Notes: pGEM^®-T Easy Vector Systems (2068)

Notes: A minigene was constructed with five exons and four introns from exons 5-9 of the CYP27 gene. The 2111bp product was generated with primers containing a start codon and a stop codon. The product was directly cloned into the pTARGET™ Mammalian expression vector and transfected into COS-1cells. RNA was isolated from these cells and RT-PCR was performed to amplify the spliced product. This assay could distinguish between normal splicing of the third intron and aberrant splicing of due to the Arg362Ser mutation. The proteins produced by the normal and mutant minigenes were also assessed by immunoblotting. (1333)

Notes: The pGEM® -T Easy Vector Systems, Wizard® PCR Preps DNA Purification System and Wizard® Plus Minipreps DNA Purification Systems were used in this study. (1959)

Notes: The S100A2 protein cDNA was subcloned into the pGEMEX^®-2 Vector and expressed in BL21(DE3)pLysS E.coli cells. The protein was purified by a referenced method. (1173)

Notes: The Access RT-PCR System was used to compare the abundance of GnT-IV mRNA in various bovine tissues. (0694)

Notes: Luciferase reporter constructs were cotransfected into 3T3-L1 preadipocytes with the pRL-CMV Vector at a 25:1 ratio and luciferase activities were determined with the Dual-Luciferase^® Reporter Assay System. RT-PCR was accomplished with AMV Reverse Transcriptase and Taq DNA Polymerase. RNase Protection Assay were performed with the aid of the RNase ONE™ Ribonuclease. (0988)

Notes: In this paper, Tfl DNA polymerase was used for two-step RT-PCR. The amplified fragments were cloned into the pGEM®-T Vector. Promega's Terminal Deoxynucleotidyl Transferase (TdT) was used to end label specific oligos for screening a frog brain cDNA library. T7 and T3 RNA polymerases were used to make digoxigenin labeled riboprobes for in situ hybridization. (1281)

Notes: Bisulphite-treated DNA was purified using the Wizard® DNA Clean-Up System prior to PCR. The amplified DNA was then cleaned using a Wizard® PCR Preps DNA Purification System prior to cloning and the cloned DNAs were sequenced using the fmol® DNA Cycle Sequencing System. (0001)

Notes: The entire coding region of the T isoform of the catalytic subunit of the acetycholinesterase (ACHET)was amplified and cloned into the pTargeT™ Vector and sequenced. The ColQ cDNA was cloned into the pTargeT™ Vector as well and mutated with a Pfu DNA polymerase-based mutagenesis system. The ACHET and ColQ mutants were coexpressed in COS cells and interactions examined with sedimentation analysis through sucrose gradients. (0595)

Notes: In this paper, a 538bp fragment was amplified and subcloned into the pGEM^®-T Easy Vector. (1483)

Notes: Streptavidin MagneSphere^® Paramagnetic Particles (SA-PMPs) were used to capture biotinylated primer probe in a random access retrieval of genetic information by PCR (rargip) screening [ABE, K., (1992) Rapid isolation of desired sequences from lone linker PCR amplified cDNA mixtures: application to identification and recovery of expressed sequences in cloned genomic DNA. Mamm. Genome 2:252-259]. The pGEM^®-T Vector System and PolyATract^® mRNA Isolation System were also used. (0513)

Notes: Genomic analysis noted a single base change between wild type and mutant. To see if this base change was enough for alternative mRNA splicing, minigenes were constructed and subcloned into the pTargeT™ Vector. The mutant and wild-type minigenes were transfected into COS-1 cells. Forty-eight hours post-transfection total RNA was isolated and analyzed by RT-PCR. Transfectants with the mutant sequence did produce a different product due to alternative splicing. (1331)

Notes: The pGEM®-5Zf(+) Vector was used for subcloning of a PCR fragment. The fragment was used to make a probe for RNase protection assays. The pGEM®-7Zf(+) Vector was used for routine subcloning of restriction fragments from the chitotriosidase gene. (1427)