...8000 (PEG) + 0.5 M NaCl Cell lysis buffer (50 mM Tris HCl, 150 mM NaCl, 2 mM MgCl2) Reagent Preparation...Aliquot and store at 4 °C. Cell Lysis Buffer : 50 mM Tris HCl, 150 mM NaCl, 2 mM MgCl 2 Add the following ... bottle: 1836 mL deionized water + 100 mL of 1 M Tris HCl pH 8.5 + 60 mL of 5 M Sodium Chloride + 4 mL...-Cap), plasmid expressing your gene of interest Triton X-100 Benzonase/DNAse I (Millipore 71205-3) 40%...tilting the CS5 back and forth. Pro-Tip To help distribute the media amongst the five layers, tilt the CS5...container. Gently pour new media into the CS5. Distribute the media evenly between the layers. Return the...PBS and add to the harvested media and cells. Centrifuge at 3900 rpm for 20 min at 4 °C to pellet the ...

...solutions: Solution I - Resuspension Buffer 25 mM Tris-HCl (pH 8) 50 mM glucose 10 mM EDTA Store Solution...towel for 5-20 min. Resuspend dry DNA with TE (10 mM Tris-HCl pH 8, 0.1 mM EDTA). Pro-Tip DNA resuspension...Last Update: Feb. 8, 2018 Equipment Desktop microcentrifuge Desktop vortexer Vacuum (optional) Reagents... (low copy plasmids require larger cultures). Centrifuge the culture to pellet the bacteria before proceeding...entire overnight culture cannot fit into a single centrifuge tube, aliquot it into several tubes/bottles. ...solution. Pellet the proteins and genomic DNA by centrifugation, and remove the plasmid-containing supernatant...the stock culture to a 1.75 mL microfuge tube. Centrifuge in microfuge tube at 10,000 g for 30 sec. Pour...

... should be resuspended in annealing buffer (10mM Tris, pH7.5-8.0, 50mM NaCL, 1mM EDTA) and mixed in equimolar... Annealed Oligo Cloning You may also like... Restriction Digest of Plasmid DNA DNA Ligation Bacterial ...this tutorial we will discuss how to add new restriction sites to the MCS of an empty vector. However,...design a set of oligos containing your desired restriction sites and add them to your existing vector. It...cloned directly into the overhangs generated by restriction digest of existing sites in the original vector...waiting for your oligos to arrive, conduct a restriction digest of 1μg of vector with EcoRI and SalI. ...

...SafeStain, Thermo Fisher, LC6060 4-12% NuPage Novex bis-tris mini gel, 1mm thick, 10-well, Invitrogen NP0321BOX... single channel pipette Pipette controller Microcentrifuge Electrophoresis chamber Power supply Rocking...VWR 89130-902 Gel loading tips, Corning 4853 Microcentrifuge tubes, Neptune 3745.X PBS, 1X pH 7.4, VWR 45000...each sample. Spin the sample briefly in the microcentrifuge. Heat the samples for 10 min at 100 °C in a...heat block. Spin the sample briefly in the microcentrifuge. Remove the gel from the plastic wrapper and...

...Sarkosyl) 0.1 M 2-mercaptoethanol TRIzol® or similar product such as TRI Reagent®, RNAzol®, QIAzol® (for...refrigerated centrifuge, you can carefully bring a centrifuge into a cold room for centrifugation. Once you...Equipment Refrigerated microcentrifuge If you only have a non-refrigerated microcentrifuge, see step 4 of either...above for the recipe). If using TRIzol®, jump down to the Option #2 - TRIzol® Protocol section below. Homogenize... -80°C. Option #2 - TRIzol® Protocol Homogenize or lyse tissues or cells in TRIzol® or a similar product...tissues: use 1 mL of TRIzol® per 100 mg of cells. For cultured cells: use 1 mL of TRIzol® per 1 X 10 7 cells...

...balanced tabletop centrifuge, with tubes evenly distributed around the rotor. How to Centrifuge Now that you...parts of a centrifuge, you’re ready to begin using the centrifuge. Determine the type of centrifuge needed...the centrifuge is balanced. Using the centrifuge in an unbalanced state can damage the centrifuge and ...features to keep in mind when using any centrifuge. However, each centrifuge may have specific instructions so...Sample to be centrifuged Water Background Information Front view of an open tabletop centrifuge with labels...found on almost any centrifuge. Types and Parts There are several types of centrifuges: Mini or Tabletop...on the centrifuge their speed ranges can vary, but are typically lower than some other centrifuge types...

...Diagnostic Restriction Digest Diagnostic Restriction Digest You may also like... Restriction Digest of ...another. A diagnostic restriction enzyme digest takes advantage of the fact that restriction enzymes cleave ...Orientation by Restriction Digest If you clone an insert into a vector by a single restriction enzyme, you...detailed protocols as well as tips and tricks see the Restriction Digest and Gel Electrophoresis pages....DNA Agarose Gel Electrophoresis Introduction Restriction enzymes are naturally occurring bacterial endonucleases...enzymes and the unique sites they recognize, restriction digests have become the most widely used method...cleave DNA at specific sequences called restrictions sites. Often, the size of the plasmid insert and vector...

... Protocols Restriction Digest of Plasmid DNA Restriction Digest...commonly used restriction enzymes, visit (Link opens in a new window) NEB . Restriction enzyme digestion...Procedure Select restriction enzymes to digest your plasmid. Pro-Tip To determine which restriction enzymes will...typical restriction digestion reaction could look like this: 1 µg DNA 1 µL of each Restriction Enzyme ...perform a restriction digest of plasmid DNA Protocols...from an Agarose Gel DNA Ligation Introduction Restriction enzyme digestion takes advantage of naturally...specific sequences. There are hundreds of different restriction enzymes, allowing scientists to target a wide...

...assist with restriction enzyme digestion (usually 3-6bp). Restriction Site: Your chosen restriction site for...fusing the upstream restriction site to the forward primer and the downstream restriction site to the reverse...product is now ready for restriction digestion. Digest Your DNA Set up restriction digests for your PCR product... Plasmid Cloning by PCR You may also like... Restriction Digest of Plasmid DNA DNA Ligation Bacterial ...of a piece of DNA and at the same time adding restriction sites to the ends of that piece of DNA so that... amplified (usually 18-21bp). When selecting restriction sites, you should use a DNA analysis tool, such...Sequence Analyzer , to allow you to identify which restriction sites are present in a given sequence. You want...

...Bio-Rad, 1863005 DG8 cartridge, Bio-Rad, 1864008 DG8 gasket, Bio-Rad, 1863009 DG8 cartridge holder, Bio-Rad...µL DMEM complete and transfer to a microcentrifuge tube. Centrifuge for 5 min at 100 x g . Gently aspirate...then spin 10 sec in a mini centrifuge and place on ice. Wipe down a DG8 cartridge holder with bleach and ...and spin in a mini centrifuge for 10 sec before use. Place an 8-well PCR tube strip into a chilled 96-... Generating the Droplets Place a DG8 cartridge into the cartridge holder. Using a 2–50 µL multichannel...Cover the cartridge with the DG8 gasket, making sure that it is secure. Transfer the cartridge holder to... Biological Safety Cabinet Aspirating unit Microcentrifuge, Eppendorf, 5425R Droplet digital PCR System...

...Plasmid Cloning by Restriction Enzyme Digest (aka Subcloning) You may also like... Restriction Digest of Plasmid...identify which restriction sites are present in a given sequence. When selecting restriction enzymes, you...desired restriction sites to flank your insert : You can use PCR Based Cloning and add restriction sites...need to avoid restriction enzymes that cut within your insert. Adding desired restriction sites to your...Information about plasmid cloning by restriction enzyme digest (subcloning), including design and experimental... another as long as it is already bounded by restriction sites that are also present in the same orientation...Backbone Reference . Background Subcloning by restriction digest is a commonly used lab technique. For ...

...buffer 2x . Add 50 µL of 1 M Trizma hydrochloride pH 9.0 to 10 microcentrifuge tubes. Cap the Gravitrap columns...Place column in centrifuge with the mark facing outward in all subsequent centrifugation step Pro-Tip Resin...4 °C Refrigerator Pipette controller Benchtop centrifuge compatible with 50 mL conical tubes NanoDrop ...89130-902 50 mL conical tubes, VWR 89039-656 Microcentrifuge tubes, VWR 89126-724 Filtering system 0.2 µm...phosphate dibasic, Millipore Sigma S7907-500G 1 M Trizma hydrochloride solution pH 9.0, Millipore Sigma ...µL fractions into the tubes containing 50 µL 1 M Trizma hydrochloride pH 9.0. Cap the tubes and vortex ...Place column in a 50 mL conical collection tube. Centrifuge column at 1000 x g for 2 min to remove storage...

...Bio-Rad, 1863005 DG8 cartridge, Bio-Rad, 1864008 DG8 gasket, Bio-Rad, 1863009 DG8 cartridge holder, Bio-Rad...then spin 10 sec in a mini centrifuge and place on ice. Wipe down a DG8 cartridge holder with bleach and ...and spin in a mini centrifuge for 10 sec before use. Place an 8-well PCR tube strip into a chilled 96-... step 9 to the NTC tube. Place a DG8 cartridge into the cartridge holder. Using a 2–50 µL multichannel...Cover the cartridge with the DG8 gasket, making sure that it is secure. Transfer the cartridge holder to...multichannel pipette Vortex, VWR, 10153-688 Mini Centrifuge, Thermo Scientific, 10199-452 Ice bucket 96-well...Bio-Rad, 1863051 8-strip PCR tubes, Axygen, PCR-02-FCP-C ddPCR 96-well PCR plates, Bio-Rad, 12001925 48-well...

...Iodixanol Gradient Ultracentrifugation AAV Purification by Iodixanol Gradient Ultracentrifugation You may also...Follow this protocol using Iodixanol gradient ultracentrifugation to get pure adeno-associated virus (AAV) ... be completed in one (long) day. Equipment Ultracentrifuge T70i rotor or equivalent Reagents Opti-Prep...-MK buffer 100X Pluronic-F68 NaCl MgCl 2 KCl Centrifugal filter units (MWCO 100 kDa) Reagent Preparation...to top off the tube. Seal the QuickSeal tubes. Centrifuge at 350,000 x g for 90 min in a T70i rotor at ... If you need more time, you can alternatively centrifuge for 2 h at 200,000 x g at 18 °C. Carefully take...#1 Prepare a row of roughly 20 open 1.5 mL microcentrifuge tubes in a rack. These will be used to collect...

...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...