...pipette Watch the Video! Centrifugation Learn about selecting and using a centrifuge to separate different... spectrophotometer Restriction Digests Cut and analyze plasmid DNA with restriction enzymes Agarose Gel...pooled-plasmid libraries Diagnostic Restriction Digest Verify a plasmid using restriction enzymes Watch the Video!...new window) Link to Video Restriction Cloning Assemble plasmids by restriction digest and ligation Cloning...Cloning by PCR Generate restriction sites by PCR Modification by Annealed Oligo Cloning Add a short stretch... Cloning (LIC) Scarless cloning with Type II restriction enzymes and T4 polymerase pLKO.1 - TRC Cloning...choice AAV Purification by Iodixanol Gradient Ultracentrifugation Purify adeno-associated virus from a preparation...

...in 1 mL of 1X PBS and transfer to a microcentrifuge tube. Centrifuge for 5 min at 100 x g . Carefully remove...Stack such that the electrical contacts are aligned with the corresponding electrical contacts on the blotting...protein from cells or tissue samples. Equipment Microcentrifuge 0.5–10 µL single channel pipette 2–20 µL single...1X PBS Lysis buffer e.g., RIPA lysis buffer Microcentrifuge tubes BCA Assay, Thermo Fisher 23227 β-mercaptoethanol...raised in a mouse. Procedure Section 1: Lyse cells Centrifuge 5 x 10 6 cells for 5 min at 100 x g . Gently ...buffer is suitable for most proteins but more stringent buffers and a sonication step may be required ... in the nucleus. Incubate on ice for 30 min . Centrifuge lysate for 15 min at 14,000 x g at 4 °C . Transfer...

...high-throughput next generation sequencing (NGS). Select a restriction enzyme for digest that will cut the shared plasmid...visualize that digestion on an agarose gel ( see restriction digest protocol here ). Lentiviral plasmids can... competition are limited. Equipment Table top centrifuge BioRad Electroporator (MicroPulser TM , Bio-Rad...Biosciences, 352059) 3X LB Agar + Antibiotic 65 mm Petri dish (VWR, 11019-552) 4 MaxiPreps (Qiagen HiSpeed... Plate 100 µL of each dilution onto a prewarmed Petri dish. Incubate plates at 30 ℃ overnight. Plate 2.5... eight bioassay plates (two plates per tube). Distribute evenly with a sterile spreader until all liquid...Maxipreps worth. Count colonies on the most dilute Petri dish. Total colony yield = count x 100 x 100 x 100...

...Diagnostic Restriction Digest Verify a plasmid using restriction enzymes Diagnostic Restriction Digest Protocol...cells AAV Purification by Iodixanol Gradient Ultracentrifugation Protocol Mulitchannel Pipetting Technique...transformation protocol and offer some tips and tricks to reduce transformation time and increase efficiency...Protocol Getting Started with Tissue Culture Tips and tricks for working with tissue culture in the lab. Blog...technique for molecular cloning, such as PCR- or restriction enzyme-based cloning. Purifying DNA from an Agarose...

...also like... Plasmid Cloning by Restriction Enzyme Digest Restriction Digest of Plasmid DNA DNA Ligation...bacteria more susceptible to either chemical or electrical based transformation, generating what are commonly...Water bath at 42 °C Ice bucket filled with ice Microcentrifuge tubes Sterile spreading device Reagents LB ... ng) into 20-50 μL of competent cells in a microcentrifuge or falcon tube. GENTLY mix by flicking the ...volume is too big, gently collect the cells by centrifugation and resuspend in a smaller volume of LB so ...

...generated by restriction enzyme digestion . Most restriction enzymes digest DNA asymmetrically across their...also like... Plasmid Cloning by Restriction Enzyme Digest Restriction Digest of Plasmid DNA Bacterial ...ligase, 5min at room temperature is enough. For trickier ligations (such as ligation of annealed oligos...

...Dilute 20 µL of Triton X-100 in 10 mL PBS. Blocking buffer: Dilute 0.5 g BSA and 30 µL Triton X-100 in 10 ... µL single channel pipette Reagents 1X PBS Microcentrifuge tubes Sterile Poly-D-lysine coated coverslips...-phenylindole (DAPI) Bovine serum albumin (BSA) Triton X-100 Primary antibody Secondary antibody Deionized...Antibody dilution buffer: Dilute 0.5 g BSA and 150 µL Triton X-100 in 50 mL PBS. 300 nM DAPI working solution...

...Cut out the 7kb band and place in a sterile microcentrifuge tube. TIP: When visualizing DNA fragments to...for plasmids that were successfully ligated by restriction enzyme digestion. However, once you have identified...use a miniprep kit to obtain DNA. 3. Conduct a restriction digest with EcoRI and NcoI: 1 μg miniprep DNA...Incubate for 20-30 minutes at room temperature. g. Retrieve HEK-293T cells from incubator. The cells should...collected during harvesting. TIP: In lieu of centrifugation, you may filter the media through a 0.45 μm...non-infringement, typicality, safety and accuracy. The distribution of any literature by Addgene is not meant to...the materials described in the literature. The distribution of materials by Addgene is not meant to carry...

...Micropipettes and tips Pipettor and pipettes Benchtop centrifuge compatible with 50 mL conical tubes Automated...DNA 500 mL vented flask 50 mL conical tubes Microcentrifuge tubes Cell counting chamber slide 30 mL luer-lock... mL of HEK293 cell suspension into a clean microcentrifuge tube. Pro-Tip Cells settle quickly and need...Transfer 10 µL of trypan blue into a clean microcentrifuge tube. Vortex the cell suspension. Transfer ...HEK293 cells and media to 50 mL conical tubes. Centrifuge for 15 min at 3100 x g to pellet the cells. Filter...

...Empty vectors for LIC typically employ type II restriction enzymes (e.g. BsaI), which cut at a specified...overhangs with a single enzyme, and remove the restriction sites from the final product (no "cloning scars...sequence and sacB counter-selection marker (see our restriction digest protocol ). After the digest is complete...corresponding to the first base removed by the restriction digest to create the free 5' end of the cut vector...

...presence of the antibiotic). Luria broth (LB) is a nutrient-rich media commonly used to culture bacteria in... are unable to digest the agar but can gather nutrition from the LB within. The addition of an antibiotic...manufacturer to make sure this is true in your case. Retrieve your molten agar mix from the autoclave. Pro-Tip...technique. Swirl the agar bottle to ensure even distribution of the antibiotic throughout the agar. Open ...

...DNA from an Agarose Gel You may also like... Restriction Digest of Plasmid DNA Agarose Gel Electrophoresis...technique for molecular cloning, such as PCR - or restriction enzyme -based cloning. Protocol: Gel Purification... the excised band, lay it down on the UV box and trim the top, bottom and sides with the razor blade. ...

...extensively. Why Gibson Cloning? No need for specific restriction sites. Join almost any two fragments regardless...least the correctly assembled antibiotic gene. This trick can also enable replacement of "inverse PCR" reactions...and screen for the correct plasmid product by restriction digest . Sequence the important regions of your...

...Electrophoresis uses an electrical field to move the negatively charged DNA through an agarose gel matrix toward a positive...Agarose Gel Electrophoresis You may also like... Restriction Digest of Plasmid DNA Purifying DNA from an Agarose...

...complementary regions Note: If you will be including a restriction site at the 5’ end of your primer, note that ...the enzyme to cleave efficiently (e.g. GCGGCG-restriction site-your sequence). Protocol Video Watch the...

...aware that some gloves may contain latex. Consider nitrile gloves as an alternative. For extra precautions...guidance is also adhered to. None of the authors, contributors, administrators, or anyone else associated with...

..., however, more expensive. With a 6 cm diameter petri plate containing solidified LB-agar, pipette 150...used for this purpose, provided that they fit your petri plate. Incubate the plate at room temperature for...

... visible in the room and on the door. BSL-2 centrifugation steps require an aerosol-tight lid and rotors...guidance is also adhered to. None of the authors, contributors, administrators, or anyone else associated with...

...polyethersulfone filter, Nalgene, 565-0010 Microcentrifuge tubes, Neptune 3745.X 10 cm tissue culture ...storage tube and store at 4 °C between harvest. Centrifuge the viral supernatant at 2100 rcf for 5 minutes...

... You may also like... Plasmid Cloning by PCR Restriction Digest of Plasmid DNA DNA Ligation Background...rate of the DNA polymerase. Deoxynucleoside Triphosphates (dNTPs): Are the building blocks added one at...

...DNase 10X DNase buffer Nuclease-free water Microcentrifuge tubes 96-well optical plate Pipette tips General...least 5 times. Seal plate with transparent film. Centrifuge at 3,000 rpm for 2 min to bring the sample to...

...pipette reagent reservoirs Pipette controller Microcentrifuge Microplate shaker ELISA plate washer (optional...clear) microplate seal, Thermo Scientific 5701 Microcentrifuge tubes, Neptune 3745.X Pipette tips, 1000 µL...

...Recovering Plasmid DNA from Bacterial Culture Restriction Digest of Plasmid DNA Background Information ...

...prepare your plasmid DNA. In the morning, when you retrieve your liquid bacterial culture, take 500 μL of ...

...also like... How to Design a Primer Diagnostic Restriction Digest Introduction Sequence verification of ...

...0.02 g to resuspend in 1 mL), you may place a microcentrifuge tube on the balance and then tare it. Then ...

...temperature, catalyze chemical reactions such as restriction digests needed in molecular biology, and thaw...

...presence of the antibiotic). Luria broth (LB) is a nutrient-rich media commonly used to culture bacteria in...

...preps, if prep was not previously filtered) Microcentrifuge tubes, Neptune 3745.X 6-well tissue culture...

...chloroquine Polyethylenimine, linear MW 25,000 Da Microcentrifuge tubes, Neptune 3745.X 10 cm tissue culture ...

...preps, if prep was not previously filtered) Microcentrifuge tubes, Neptune 3745.X 6-well tissue culture...

...using to hold the measured liquid (ex: another microcentrifuge tube, a bottle etc.). If there is already other...

...preps, if prep was not previously filtered) Microcentrifuge tubes, Neptune 3745.X 6-well tissue culture...

... filtration through a 0.45 µm PES filter or centrifugation at >500 x g for 5 minutes. Do not use the medium...