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reliable cDNA synthesis of a full range of ..
Polymerase chain reaction (PCR) is extensively applied in gene cloning. But due to the existence of introns, low copy number of particular genes and high complexity of the eukaryotic genome, it is usually impossible to amplify and clone a gene as a full-length sequence directly from the genome by ordinary PCR based techniques. Cloning of cDNA instead of genomic DNA involves multiple steps: harvest of tissues that express the gene of interest, RNA isolation, cDNA synthesis (reverse transcription), and PCR amplification. To simplify the cloning procedures and avoid the problems caused by ubiquitously distributed durable RNases, we have developed a novel strategy allowing the cloning of any cDNA or open reading frame (ORF) with wild type sequence in any spliced form from a single genomic DNA preparation.
Taq DNA polymerase, T4 DNA ligase, DL2000 DNA marker, Blood Genome DNA Extraction Kit, and restriction endonucleases were purchased from Takara Biotech Co., Ltd. (Dalian, China). Phusion high-fidelity DNA polymerase and T7 endonuclease I were from New England Biolabs, Inc. (Ipswich, MA). IPTG, X-Gal, and other chemicals were from Sigma-Aldrich, Inc. (St Louis, MO). Oligonucleotides were synthesized and purified by polyacrylamide gel electrophoresis by AuGCT Biotech Co., Ltd. (Beijing, China). TA cloning vector (pGEM®-T easy vector) was purchased from Promega Biotech Co., Ltd (Madison, WI). DH5α competent cells were from Tiangen Biotech Co., Ltd (Beijing, China). Glass milk DNA purification kits were from BioDev-Tech Co., Ltd (Beijing, China).
Universal RiboClone® cDNA Synthesis System Protocol - Promega
One advantage of the cDNA cloning strategy described in this report is that neither RNA extraction nor reverse transcription is needed, and thus it saves the costs of RNA extraction kits and cDNA synthesis kits. The second advantage is that it is not necessary to handle any specific tissues for a particular gene and the expression level of the gene is not a problem. The third advantage is that high fidelity DNA sequence is obtained with high efficiency while the process is simple and easy. The entire assembly process takes only a few hours. We found that even if some of the first round PCRs were not very successful (PCR product bands were not visible in ethidium bromide stained agarose gel), it usually did not affect the final full-length DNA amplification. So it is usually not necessary to optimize PCR conditions even if one or more exon primer pairs do not work very well in the first round PCR.
The Protein Structure Initiative (PSI), funded by the US National Institutes of Health (NIH), provides a framework for the development and systematic evaluation of methods to solve protein structures. Although the PSI and other structural genomics efforts around the world have led to the solution of many new protein structures as well as the development of new methods, methodological bottlenecks still exist and are being addressed in this 'production phase' of PSI. The PSI has charged all centers to participate in a plan to “store and distribute PSI clones generated by the PSI Centers.” While the is a separately funded activity, CESG has allocated significant resources in developing a plan to fulfill the important mission of transferring clones and plasmids to the PSI-MR. Issues ranging from intellectual property rights to tactical sample handling, materials verification, inter-site communication protocols, and permanent data storage have been are being addressed to facilitate the transfer, verification, tracking, and distribution of expression clones and other materials. A preliminary success rate for transfer of eukaryotic expression clones has been established, and other PSI-derived expression vectors and materials have been deposited and are becoming available for distribution. Although our center incurred substantial costs in establishing this transfer pipeline, we foresee that we will benefit from the long-term savings resulting from eliminating the need for the individual, customized materials transfer agreements and internal preparations that were previously required prior to shipping CESG’s clones and vectors to requestors around the globe. Ongoing challenges associated with transfer of larger numbers of expression clones will need to be addressed.
First Strand cDNA Synthesis Protocols (E6300) | NEB
Because fixation is so critical for good ultrastructure, we present below several alternative protocols for TEM. At the end of the section we present a method for scanning EM (SEM) of worms. In this method, whole-mount animals or structures are viewed by EM, offering both broad-scale and highly resolved images ().
Gene cloning is one of the most frequently used technologies in a molecular biology laboratory. To study a particular gene, the first step is usually to clone and express it. However, most of the eukaryotic genes are interrupted by intervening sequences (introns), which make the gene of interest very large. Manipulation of the large genomic DNA is tedious and problematic due to size capacity of cloning vectors and multiple restriction endonucleases which make it difficult to find appropriate enzymes for subcloning. To circumvent these difficulties, the cDNA is often used instead of its large genomic counterpart. But cDNA cloning is usually troublesome, which involves mRNA preparation and reverse transcription, and thus requires RNA extraction kits and reverse transcription kits. For a particular cDNA cloning, a specific tissue with a relatively high level expression is often needed for the mRNA or total RNA extraction. Some of these tissues are quite rare and difficult to obtain, and some of the genes only have a very low level expression. Furthermore, RNases are ubiquitously expressed in tissues and RNase products are commonly used in molecular biology protocols (such as plasmid DNA preparation). Since RNases are very stable and difficult to remove completely, extra care must be taken when working with RNA. Lastly, RNAs are unstable polynucleotides and thus present their own problems in handling and storage.
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First Strand cDNA Synthesis Protocols ..
Immobilized ligands (such as HaloLink™ Resin) can be used to precipitate HaloTag® fusion proteins cross-linked to DNA. This protocol abolishes the need for antibodies in classic chromatin immunoprecipitation (ChIP) assays and enables experiments in the absence of highly functional antibodies. Please refer to Promega’s manual #TM075 for this application.
Typical cDNA Synthesis Protocol;
Particular cell types can be labeled with GFP, sorted using fluorescence activated cell sorting (FACS), and studied as a homogeneous population (e.g. to examine expression using gene arrays). Furthermore, RNA interference can be used to inactivate gene expression in cultured cells, allowing for a quasi-genetic dissection of processes in culture. Finally, cells in culture are readily accessible to electrophysiological studies. Methods for culturing embryonic cells were recently developed (; ) and protocols are presented below (). The procedure has been mostly tried with embryonic cells. In general, differentiation of cells in culture seems to proceed through what would be the L1 stage. Markers specific for later stages are not expressed.
cDNA Protocols | Thermo Fisher Scientific
The “Genomic DNA Splicing” protocol avoids RNA preparation and reverse transcription steps, and the entire assembly process can be finished within hours. Since genomic DNA is more stable than RNA, it may be a more practical cloning strategy for many genes, especially the ones that are very large and difficult to generate a full length cDNA using oligo-dT primed reverse transcription. With this technique, we successfully cloned the full-length wild type coding sequence of human polymeric immunoglobulin receptor, which is 2295 bp in length and composed of 10 exons.
A collection of CDNA Protocols for research, ..
Remove supernatant. For preparations that will be sorted immediately to isolate GFP-labeled cells, resuspend the pellet in egg buffer (~107 cells/ml; See FACS protocol, Section D. below). For cells that will be maintained in culture, resuspend pellet in L15-10 media (~107 cells/ml). (See cell culture protocol, Section C. below.)
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