Electroporation competent cell protocol
[DOCX File]Addgene
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(The cell should be the host cell, where you want to do your screen ) Electroporation protocol: Add ~ 50ng of plasmid (maximum 2ul) to the bottom of a cold 1.5 ml Eppendorf tube. Add 20 ul of thawed electro-competent cells. Transfer 20 µL of the bacteria/plasmid mixture into 1 …
[DOC File]AGROBACTERIUM ELECTROPORATION COMPETENT CELLS
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AGROBACTERIUM ELECTROPORATION COMPETENT CELLS (from Steve Farrand’s lab, 1996.) 1. Grow cells overnight in 5 ml MGL medium at 28 °C with aeration. 2. Dilute the overnight culture 1/100th in 100 ml MGL in a 1L flask and. continue the incubation until OD600 ≈ 0.5 (Check OD600 after 2 hours. and thereafter every 15-20 minutes). 3.
[DOC File]Preparation of electrocompetent, recombination-induced ...
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Recombination-competent cells were prepared and transformed with ~100 ng of double-stranded oligo with the homology arms and the SNP base pair. After electroporation and recovery, 100 µl of a 1:100 dilution of cells were plated on LB + choloramphenicol (12.5 µg/ml) + streptomycin (5 mg/ml).
[DOCX File]TABLE OF CONTENTS - University of Kentucky
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Competent Cell Preparation. This procedure makes . E. coli. receptive to DNA mediated transformation by the calcium chloride method. CaCl. 2 treatment and alternative approach to competent cell preparation and transformation (electroporation) is discussed in PGMG pages 24-25. For Each Group. A fresh M9 (minimal medium plus biotin) plate of . E ...
[DOC File]Electro-competent
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The Preparation of E. coli (BJ5183) by Electroporation(06-24-2005 wyy) Place the competent cells on LB plate, 37°C overnight (16-20h). Pick a single bacterial colony from a plate that has been incubated for 16-20 hours at 37°C. Transfer the colony into 20 ml of LB medium in a 100-ml flask.
[DOC File]PROTOCOLS FOR RECOMBINANT DNA ISOLATION, …
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There are two main methods for preparation of competent bacterial cells (14) for transformation, the calcium chloride and the electroporation method. For the calcium chloride method, a glycerol cell culture stock of the respective E. coli strain is thawed and added to 50 ml of liquid media.
[DOCX File]TABLE OF CONTENTS - University of Kentucky
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Competent Cell Preparation. This procedure makes . E. coli. ... 1999) suggests that a minor modification of the electroporation protocol that includes a tRNA assisted ethanol precipitation of the ligated DNA can increase plasmid transformation efficiencies by 400-fold) Here is a link to . multiple programs for. RNA structure prediction ...
[DOC File]ELECTROPORATION OF A
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ELECTROPORATION OF A. TUMEFACIENS CELLS WITH PLASMID DNA (Protocol from Steve Farrand’s lab, 1996). 1. Thaw electrocompetent cells on ice. 2. Incubate 80µl competent cells with 1µl (50-250ng) plasmid DNA which is in water, 10 mM TRIS, or TE.
[DOC File]An effective strategy to confer electroporation competence ...
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With the appropriate selection marker, the described electroporation protocol gave high transformation efficiencies, even for large theta replicating plasmids with low copy number per cell (Table 2). Interesting results were also obtained with two other strains of B. cereus (6X106 ufc µg-1 ml-1 with HER-1399 and 3X106 ufc µg-1 ml-1 with HER ...
[DOC File]DNA digestion and Size Selection - University of Sheffield
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To check the electroporation is working and the competent cells are competent (ie can be transformed) Record the amount of bluescript used (10ng is enough) in order to calculate the efficiency of the cells to take up DNA. Ensure the plasmid used (PUC19, Bluescript , etc) carries ampicillin resistance. Use 0.1 - …
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