... Protocols Sequence Analysis Sequence Analysis of a Plasmid You ...performing sequence analysis of a plasmid Protocols...

... carry one or more antibiotic resistance genes, which confer resistance to a specific antibiotic to the...to be resistant to the antibiotic. On the second plate, streak out a strain that’s not resistant to the... be resistant to the antibiotic, (+) indicates that the tested strain is supposed to be resistant to the...Negative Result 3: Only the Non-resistant Strain Grows If only the non-resistant strain grows, it’s likely ... carrying them. The presence of an antibiotic resistance gene on a plasmid allows researchers to easily...for the selection of only those bacteria with resistance to that antibiotic - usually conferred by a plasmid... plasmid carrying the antibiotic resistance gene. The following protocol will allow you to make your own...

... Protocols Agarose Gel Electrophoresis Agarose Gel Electrophoresis You may also like... Restriction...Standard protocol for performing agarose gel electrophoresis, including tips to improve resolution and separation... Agarose Gel DNA Ligation Introduction Gel electrophoresis is the standard lab procedure for separating...pairs) for visualization and purification. Electrophoresis uses an electrical field to move the negatively...protocol video below to learn how to perform gel electrophoresis. Equipment Casting tray Well combs Voltage ...solidified, place the agarose gel into the gel box (electrophoresis unit). Fill gel box with 1xTAE (or TBE) until... short DNA fragments is polyacrylamide gel electrophoresis (PAGE), which is typically used to separate...

...became automated with the discovery of a heat resistant DNA polymerase from the thermophilic bacterium...several cycles of denaturation, annealing, and synthesis. After the PCR is complete, the product can be...be verified based on size by gel electrophoresis . Protocol Video Watch the protocol video below to learn...temperature step-wise by 1-2°C. The rate of DNA synthesis is ~1-2 kb/min. The extension time can be adjusted...of DNA that we are interested in amplifying for analysis and manipulation. It is essential that the template...cations can also be used for PCR-mediated DNA mutagenesis. A higher cation concentration increases the ...that are starting points for DNA elongation and synthesis. Taq DNA polymerase: Special DNA polymerase that...

...your restriction digest results. Equipment Electrophoresis chamber Pipetman Reagents Liquid DNA aliquot...manufacturer's instructions) Gel loading dye Electrophoresis buffer Pipet tips Procedure Select restriction...sequence (and where they will cut), use a sequence analysis program such as Addgene's Sequence Analyzer . ...visualize the results of your digest, conduct gel electrophoresis . Tips and FAQ Restriction enzymes MUST be ...Dam or Dcm methylase positive strains will be resistant to cleavage at certain restriction sites. See ...diagnostic digest), you can create a "Master Mix" consisting of all of the reaction components except for ...each tube. This will save you time and ensure consistency across the reactions....

...separated by denaturing polyacrylamide gel electrophoresis alongside serial dilutions of a standard antibody...Run SDS-PAGE and stain gel Day 1 or later: Image analysis Video Watch this instructional video to learn ...pipette Pipette controller Microcentrifuge Electrophoresis chamber Power supply Rocking platform Fume ...the bottom of the gel. Place the gel in the electrophoresis chamber and secure it. Note: Check the manufacturer...make quantifying the gel easier. Cover the electrophoresis chamber and attach to a power supply. Run the.... Turn off the power supply and unplug the electrophoresis chamber. Remove the gel from the chamber. Use...Addgene QC and would not be used. Section 3: Image Analysis Use ImageJ (Link opens in a new window) or a similar...

...imager -80 °C freezer Reagents 1X PBS Lysis buffer e.g., RIPA lysis buffer Microcentrifuge tubes BCA Assay...in appropriate volume of cold lysis buffer. Pro-Tips The volume of lysis buffer will vary depending on...will generally be between 250–1000 µL . The ideal lysis buffer will vary depending on the cellular location...the bands. Tips and Troubleshooting The optimal lysis buffer will vary depending on the sample type and...protein of interest. You may need to try a variety of lysis buffers to find the best for your target. The optimal...

...Restriction Digest of Plasmid DNA Agarose Gel Electrophoresis Introduction Restriction enzymes are naturally...and analyze the resulting fragments by gel electrophoresis . The pattern of the fragments on the gel can...enzyme or enzymes that cut your plasmid. Many DNA analysis tools, including Addgene’s Sequence Analyzer ,...to analyze a restriction digest. Equipment Electrophoresis chamber Pipetman Pipet tips Reagents Liquid...manufacturer's instructions) Gel loading dye Electrophoresis buffer Verifying Total Plasmid Size -OR- Insert... tricks see the Restriction Digest and Gel Electrophoresis pages....

... for Ampicillin resistance but is much more important for other antibiotic resistances. Plate some or ...bacterial origin of replication and an antibiotic resistance gene for use as a selectable marker in bacteria... the bacteria time to generate the antibiotic resistance proteins encoded in the plasmid backbone so that...Shorten or skip the outgrowth (for Ampicillin resistance it is ok to completely skip the outgrowth, for... plate containing the correct antibiotic. The resistance gene on your plasmid must match the antibiotic...

... Technology Protocol Agarose Gel Electrophoresis Gel electrophoresis is the standard laboratory procedure... once using a multichannel pipette and avoid inconsistent pipetting with our simple tips Bacterial Transformations...visualization and purification. Agarose Gel Electrophoresis Protocol Purifying DNA from an Agarose Gel ...DNA fragments based on size. Following gel electrophoresis, you can cut DNA bands out of the agarose gel...

...gel electrophoresis, which separates DNA by their length in base pairs. Following electrophoresis, you...Restriction Digest of Plasmid DNA Agarose Gel Electrophoresis DNA Ligation Background Information Gel purification...Protocol: Gel Purification Follow the Agarose Gel Electrophoresis Protocol with the following amendments: Notes...

... carry one or more antibiotic resistance genes, which confer resistance to a specific antibiotic to the... carrying them. The presence of an antibiotic resistance gene on a plasmids allows researchers to easily...antibiotic in your LB media matches the antibiotic resistance on your plasmid. If the bacteria on your LB agar...

...the Video! Sequence Analysis Verify important plasmid features using sequence analysis RNA Extraction Without...plasmid DNA with restriction enzymes Agarose Gel Electrophoresis Separate DNA by size on an agarose gel Watch...Lentivirus Quantify virus by counting antibiotic resistant colonies Generating Stable Cell Lines with Lentivirus...

...plasmids by designing primers to split an antibiotic resistance gene to effectively create an extra part, one... Assembly master mix (isothermal reaction mix) consists of three different enzymes within a single buffer...Assembly® is licensed to New England Biolabs by TelesisBio. Gibson DG, Young L, Chuang RY, Venter JC, Hutchison...Hutchison CA, Venter JC, Merryman C. (2010). Chemical synthesis of the mouse mitochondrial genome. Nature Methods...

...lentiviral preparations that confer antibiotic resistance....lentivirus preparations that confer antibiotic resistance. Determining the titer of your lentiviral preparation...selection markers. Note this assay requires staining resistant colonies with crystal violet solution and, therefore...media as needed Days 14–18: Stain cells and count resistant colonies Equipment Class II, Type A2 Biological...

...Timeline Plate set-up: 2 h qPCR run: 1.5 h Data analysis: 30 min Equipment qPCR instrument Heating plate... #59462 is one plasmid that gave reliable and consistent results. Use the recommended plasmid, or test... Sample 4 F E Sample 2 Sample 5 F Perform data analysis using the instrument’s software. Determine the...determine differences between samples. Melt curve analysis: a single peak should be seen. The presence of...

...Polyethylene Glycol 8000 (PEG) + 0.5 M NaCl Cell lysis buffer (50 mM Tris HCl, 150 mM NaCl, 2 mM MgCl2)...separate into phases. Aliquot and store at 4 °C. Cell Lysis Buffer : 50 mM Tris HCl, 150 mM NaCl, 2 mM MgCl...resuspend the pellets in a total of 5 mL of cell lysis buffer (recipe above). Pipet back and forth to resuspend... the cells by adding a total of 12.5 mL of cell lysis buffer. Pipet back and forth to resuspend each pellet...

...target cell line. Once introduced, the puromycin resistance marker encoded in pLKO.1 allows for convenient...drives expression of puromycin. Puro R Puromycin resistance gene for selection of pLKO.1 plasmid in mammalian...bacterial origin of replication. Amp R Ampicillin resistance gene for selection of pLKO.1 plasmid in bacterial...allow for sufficient expression of the puromycin resistance gene. f. To select for infected cells, add puromycin...you as a guide and on an “AS IS” “AS AVAILABLE” basis without warranty of any kind either expressed or...

...containing the carbenicillin (amp) resistance gene (or other antibiotic resistance) Procedure Day 1 Prepare carbenicillin... cells containing plasmids differing in their resistance genes, as one does not need to prepare separate...

...like... Bacterial Transformation Agarose Gel Electrophoresis Agarose Gel DNA Purification Streaking and ...purity click here . Top Protocol: Kit-free Alkaline Lysis Plasmid Miniprep Prepare the following solutions...phase Middle phase - A white layer may appear, consisting of precipitated protein particles Bottom phase...