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1、菌落pcr和质粒pcr对转化菌的筛选（Screening of transformants by colony PCR and plasmid PCR）Colony PCR and plasmid PCR can be used to screen transformants.txt can hold up and put it down. Its called weight lifting. If you can hold it up and put it down, it is called weight lifting. The head should have courage, and
2、 rise to have the spirit. Learn to add, pride to subtract, opportunity to ride, sloth to divide. The three problems of life: thinking, love, Unrequited love.Screening of transformants by colony PCR and plasmid PCRArticle source: 2006-7-19 10:16:32Screening of transformants by colony PCR and plasmid
3、PCRJournal of immunology, 2000, issue second, sixteenth volume, techniques and methodsShen cares about Zhu Huifen, Zhang Yue, Wang Shuo, Zhu Zhigang and Zhou ChunSetting: Department of immunology, Tongji Medical University, Hubei, Wuhan 430030, ChinaKeywords: colony PCR; plasmid PCR; sequence analys
4、isAbstract: Objective To establish a simple and reliable method for screening transgenic bacteria. Methods colony PCR and recombinant plasmid PCR were used to screen the transformants and analyze the DNA sequences. To select and identify the cloning vectors and expression vectors, a universal primer
5、 or an immunoglobulin family specific primer complementary to the vectors, inserted into a five point sequence, was selected and identified. Positive colonies and plasmid PCR products were sequenced automatically by enzyme digestion as templates. Results colony PCR and plasmid PCR technology could b
6、e used as gene cloning, screening and identification methods. Conclusion colony PCR and plasmid PCR were simple, fast and reliable, and their products could be used as template for sequence analysis.Classification number: Q78; Q782; Q783; document identification code: AArticle number: 1090-8861 (200
7、0) 02-0151-03Sereening, of, transfected, bacteria, by, bacteria, colonies, PCR, and, plas-mid, PCRSHEN, Guan-xin, ZHU, Hui-fen, ZHANG, Yue, WANG, Shou, ZHU, Chun (Department, of, Immunology, Tongji, Medical, University, Wuhan430030, China)Abstract:Objective we have developed a procedure that allows
8、rapid screening of transfected bacteria colonies and di-rect sequencing of bacteria colonies as well as plasmid pCR-products.Methods in the first step the multiple cloning sites con-taining the sequences of interest are amplified by bacteria colonies PCR and plasmid PCR using lacZ specific primers i
9、n cloned vectors.For screening of expression vectors of VH and VL of monoclonal antibodies we have used family specific primers.Colonies pCR-and plasmid PCR products are purified ENZYMATICALY and are used as templates in dye-primer or dye-terminator sequencing.Results The procedure allows fast seque
10、ncing of a large numbers of samples with minimal on-hands time and minimal need For precipitation/centrifugation steps.Using this method, we routinely can read 600 or more nucleotides for on a standard aBI 120 PCR-products 373A sequencer.Conclusions despite of the fact that our methods requires an a
11、dditional PCR-step, its faster, cheaper and more reliable than the standard method using highly puuified plasmid DNA.Keywords:bacteria colonies; PCR; plasmid pCR; sequencesPCR products are directly sequenced as a model, and they are fast, simple and convenient. In this study, the universal primers (
12、LacZfor and LacZback) complementary to the inserted ends of the vectors were used, and the clones were screened and identified by cloning vector.The target gene 5end and 3 end specific primers were used for screening and identification of vector transfected bacteria. 9 sequences of monoclonal antibo
13、dies, variable region genes, 120 positive plasmids PCR and colony PCR products were sequenced as DNA sequencing templates, and the following reports were reported as follows. Colonies or plasmid PCR primers of 1.2.1 cloning vector: synthesized by PerkinElmer company.LacZback:5 -GCTTCCGGCTCGTATGTTGTG
14、TGG-3; LacZfor:5-CGCCAGGGTTTTCCCAGTCACGACG - 3.1.2.2 expression vector of colony or plasmid PCR primers: mouse immunoglobulin family specific primers 1 5, synthesized by PerkinElmer company.1.2.3 sequencing primers: M13 / pUC (-29) back:5-CAGGAAACAGCTATGAC-3; M13 / pUC (-40) for:5 -GTTTTCCCAGTCACGAC
15、-3 ; 1.3 Plasmid Extraction Reagent QIAprepPlasmidMiniPrepKit purchased from Qiagen company.1.4 transformed recipient bacteria transformed target genes into JM109 competent bacteria.1.5 transformant screening 1.5.1 colony PCR: with a sterile toothpick respectively from single white colonies, soaked
16、in liquid containing 20 l0.1%Triton - 100 C heating 2min. 2 l was used as template, and LacZfor and LacZback as primers were used for PCR amplification. 10 lPCR products were prepared by 2% agarose gel electrophoresis and subjected to UV light transmission.Screening and identification of VH and VL expressing vectors: the same method was used for colony processing, taking 2 mu l as template,
