MBg-1 Introduction.pptVIP

Practical skills will be provided in our course, such as: Tips for gene cloning (most asked questions) Dephosphorylation is sometimes necessary to prevent self ligation, but for ligation to occur, at least one of the DNA ends (insert or vector) should contain a 5′ phosphate. Primers are usually supplied non-phosphorylated; therefore, the PCR product will not contain a 5′ phosphate. Digestion of DNA with a restriction enzyme will always produce a 5′ phosphate. A DNA fragment can be phosphorylated by incubation with T4 Polynucleotide Kinase. Addition of 6 bases upstream of the restriction site is sufficient for digestion with most enzymes. When visualizing DNA bands with a transilluminator, you should not use short wavelength UV light of 312 nm or below because the DNA will be severely damaged. Use 360-365 nm UV light instead to minimize UV exposure that may cause DNA damage. 请把你的问题告诉我。 Reference measurements along the left side marks a 10-fold decrease in size Most cells are between 1 and 100um in diameter DNA Replication Operon 操纵子? （启动基因＋操纵基因＋结构基因） R-PCR建立的背景及应用 目前，研究基因差异表达谱主要是通过高通量二代测序（RNA-seq），生物芯片(microarrays)，抑制差减杂交（Subtractive Suppression Hybridization，SSH）等完成的。这些方法各有优缺点，且没有一种方法可以简易高效地从基因群体中去除非目标基因。PCR及限制性酶切都是比较可靠高效的技术。限制性酶ApeKI是NEB公司的一种 time-saver 酶且能与PCR反应缓冲液兼容。根据这些技术特点我们建立了R-PCR技术。本技术可应用于研究差异表达的基因及特异性的基因组DNA，从而克隆目标基因。R-PCR可替代的技术包括抑制差减杂交（SSH），差异显示（DD）等。R-PCR产物可自制成芯片用于作图等。 基因消除 Removing PCR 基因消除PCR是基于限制性酶的PCR反向过程的一种去除非目标基因的新技术，简称为R-PCR (Removing PCR, Restriction PCR, or Reverse PCR)。在PCR反应中，每一循环目标基因得到了扩增；在R-PCR反应中，每一循环非目标基因得到了去除。 如果你想扩增你想要的基因，你可用PCR；如果你想去除你不想要的基因，你可用R-PCR。 Removing PCR for the elimination of undesired DNA fragments cycle by cycle A novel removing polymerase chain reaction (R-PCR) technique was developed, which can eliminate undesired genes, cycle by cycle, with efficiencies of 60.9% (cDNAs), 73.6% (genomic DNAs), and ～100% (four DNA fragments were tested). The R-PCR is an easy and cost-efficient met