... Protocols DNA Quantification DNA Quantification You may also like... Addgene’s DNA Quantification Protocol. Protocols... to protein (260/280) is generally used as an indicator of the purity of DNA samples. These days, many...the NanoDrop, if two consecutive samples have significantly different concentrations, it is possible that...between samples if they are expected to vary significantly in concentration. DNA dissolved in water is ...

...Kit (one conical is its own Maxiprep). Critical Do not freeze pellets for later purification. Immediately... Protocols CRISPR Library Amplification CRISPR Library Amplification You may also like... Pooled libraries...quantities of library for experimental applications. Repeated amplifications should be avoided as best as possible...origin of replication and antibiotic selection). This recombination, at a low rate, is not typically a problem...Follow this protocol to perform amplification of CRISPR pooled plasmid libraries in Escherichia coli ...refer to our pooled library material pages for amplification protocols that have been developed by the depositor...available. If a pooled library does not yet have an amplification protocol, the following protocol can be used...

...ultracentrifugation. The arrow indicates the 60–40% interface. The vertical black line indicates the location of the... Protocols AAV Purification by Iodixanol Gradient Ultracentrifugation AAV Purification by Iodixanol Gradient...isomolar density gradient medium suitable for virus purification and isolation of cells, organelles, lipoproteins...and use an iodixanol column gradient for AAV purification. Workflow Timeline Day 1: Purify Day 2: Buffer... to collect the solution, or you will lose a significant amount of your virus. When the first fraction..., et al. "Recombinant adeno-associated virus purification using novel methods improves infectious titer...

... Protocols Plasmid Modification by Annealed Oligo Cloning Plasmid Modification by Annealed Oligo Cloning...added when ordering them or can be performed enzymatically later. Experimental Procedure Digest and purify...

...Affinity Purification with Protein A or Protein G Recombinant Antibody Affinity Purification with Protein...: Buffer exchange Equipment Class II, Type A2 Biological Safety Cabinet 4 °C Refrigerator Pipette controller...controller Benchtop centrifuge compatible with 50 mL conical tubes NanoDrop spectrophotometer 37 °C, 5% CO 2...VWR 89130-900 50 mL pipette, VWR 89130-902 50 mL conical tubes, VWR 89039-656 Microcentrifuge tubes, VWR...inhibitors to 1X if this has not already been done. We typically start with about 250 mL of supernatant and add...buffer. re Collect the flow-through in a 50 mL conical tube and dispose after use. Carefully pour the ...closure and loosen cap. Place column in a 50 mL conical collection tube. Centrifuge column at 1000 x g ...

...Vector by Gel Purification Run your digest DNA on an agarose gel and conduct a gel purification to isolate...analyzing the gene’s function. The process is shown graphically in the following cartoon, in which we are adding...amplify and design primers that will bind to and replicate it. The following image shows the ends of the ...need to use the reverse complement to get PCR amplification. We can start similarly, taking the final 18bases...the (Link opens in a new window) QIAquick PCR Purification Kit . The PCR product is now ready for restriction...plasmid. Because you lose some DNA during the gel purification step, it is important to digest plenty of starting...reaction and 1μg of recipient plasmid. It is also critical that as much of the recipient plasmid as possible...

...10x magnification. To determine the final magnification of your image, multiply the magnification of your...lab! Introduction Microscopes are emblematic of biological research and are found in many different types...suggests, light microscopes take advantage of the physical properties of light to detect small objects. Two...most important properties of a microscope are magnification (the ability to make an image larger) and resolution...reaches your eyes. These lenses determine the magnification of the image and the resolution your microscope...information, but the most important (for now) is the magnification power, such as 4x, 10x, or 20x. A 4x objective...times, and so on. The ocular lens also provides magnification and the power should be provided on the microscope...

...page you'll find practical lab protocols that you can use for a wide range of applications, with videos for...Video! DNA Purification Miniprep, phenol-chloroform extract, and precipitate DNA DNA Quantification Measure...protocols are the building blocks for many more complicated procedures. Plasmid Cloning Protocols for constructing...preparations. Antibodies Protocols for common antibody applications. Intro to the Lab Bench Name Description (Link...bacterial strain Watch the Video! CRISPR Library Amplification Amplify CRISPR pooled-plasmid libraries Diagnostic...Cloning by PCR Generate restriction sites by PCR Modification by Annealed Oligo Cloning Add a short stretch...Generating Stable Cell Lines with Lentivirus Genomically integrate your DNA sequence into a cell line ...

...Load 5 μL of each standard in duplicate Load 5 μL of each sample in duplicate. Do not forget to include a...dilution is appropriate). Quality of duplicates: Exclude duplicates from analyses if there is more than...Nuclease-free water Microcentrifuge tubes 96-well optical plate Pipette tips General Considerations Always...Always run standards and samples in duplicate at least If possible, include an AAV reference sample of known...should also contain an internal passive reference (typically ROX dye), to normalize non-PCR–related fluorescence...vortex) Incubate 30 min at 37 °C Transfer to ice ** Critical: do NOT treat your plasmid standard with DNase...DNase ** Make 6 serial dilutions, in duplicate, of your standard curve plasmid (2 x 10 9 stock made in step...

...vector by gel purification Run your digested DNA on an agarose gel and conduct a gel purification to isolate...treatment of your recipient plasmid as well as a specifically designed test digest later to verify that the...plasmids. Because you lose some DNA during the gel purification step, it is important to digest plenty of starting...plasmid and 1μg of recipient plasmid. It is also critical that as much of the recipient plasmid as possible...prior to the ligation step or prior to the gel purification step, depending on the phosphatase you choose...isolate the DNA. When running a gel for purification purposes it is important to have nice crisp bands and...bands away from the gel via your favorite gel purification method, it is important to determine the concentration...

... of sonication to avoid overheating of the sample. Mix well between rounds of sonication. Sonicate until...am): Harvest cells Equipment Class II, Type A2 Biological Safety Cabinet 0.5–10 µL single channel pipette...container pH meter Stir plate Magnetic stir bar Sonicator Ear protection Vortex Reagents Adherent HEK293T...pyruvate, Corning 10-014-CV (optional) 7.5% sodium bicarbonate, Corning 25-035-CI (optional) 1 M HEPES, HyClone... 7.5 mL 100X glutaGRO, 7.5 mL of 7.5% Sodium Bicarbonate, 7.5 mL 1 M HEPES to 750 mL DMEM + 1 g/L glucose...Before You Start The health of the HEK293T cells is critical for optimal AAV yield. Do not overgrow your cells... layers are covered with media. 293T cells are delicate and detach very easily - media should always be...

...Complete to a 15 mL conical and an appropriate amount of 25 mM chloroquine to the conical so that the final...lentivirus can be used for a variety of downstream applications such as stable-cell line generation. Workflow...am): Harvest virus Equipment Class II, Type A2 Biological Safety Cabinet 0.5–10 µL single channel pipette...10 cm tissue culture dish, Corning 430167 15 mL conical tubes, VWR 21008-216 Hydrochloric acid Sodium hydroxide... or sodium hydroxide to adjust the pH to 7.0. Typically, the solution will be basic and will need adjustment...optimal mass DNA:mass PEI ratio will need to be empirically determined for each new batch of 1 mg/mL PEI ...Start The health of the packaging cell line is critical for obtaining high viral titer. 293T cells should...

...PEI may vary significantly and should be empirically determined for your sample. Typical ratios may range...General Transfection Protocol Recombinant Antibody Purification Protocol Introduction Transfections allow for...antibody can be purified for use in a variety of applications. Sharing speeds science. We believe that sharing...antibody-expressing plasmids containing the SV40 origin of replication use a HEK293 line stably expressing the SV40 ... containing the Epstein-Barr virus origin of replication use a HEK293 line stable expressing Epstein Barr...pipettes Benchtop centrifuge compatible with 50 mL conical tubes Automated cell counter 37 °C, 5% CO 2 incubator...antibody plasmid DNA 500 mL vented flask 50 mL conical tubes Microcentrifuge tubes Cell counting chamber...

... all publications arising from the use of this vector. A.2 Map of pLKO.1 pLKO.1 is a replication-incompetent...Sigma #A9414 Luria Broth Agar (LB agar) American Bioanalytical: #AB01200-02000 Ampicillin VWR: #7177-48... antibiotic Per 40 grams of powder from American Bioanalytical catalog # AB01200-02000, LB contains: 10g...terminal repeat. f1 ori f1 bacterial origin of replication. Amp R Ampicillin resistance gene for selection...bacterial cells pUC ori pUC bacterial origin of replication. 5’LTR 5’ long terminal repeat. RRE Rev response...gene. For example, the Whitehead Institute for Biomedical Research hosts an siRNA Selection Program that... Sigma-Aldrich: #H9268 Protamine Sulfate* MP Biomedicals: #194729 Puromycin* Sigma-Aldrich: #P8833 Target...

...Stack such that the electrical contacts are aligned with the corresponding electrical contacts on the blotting...Immunocytochemistry Protocol Recombinant Antibody Purification Protocol Introduction Western blot is a technique...most proteins but more stringent buffers and a sonication step may be required for hard to extract proteins...BSA standard that range from 0–2000 µg/mL . In duplicate, dilute 10 µL of standard, blank, and lysate samples... nm . Calculate the average absorbance of the duplicate samples on the plate. Subtract the average absorbance...vary between samples and target proteins but is typically between 10–50 µg . If the protein is in low abundance...Bottom Stack on the blotting surface. Align the electrical contacts on the blotting surface of the iBlot...

...make bacteria more susceptible to either chemical or electrical based transformation, generating what are...bacteria are used as the means for both storing and replicating plasmids. Because of this, nearly all plasmids...expression) carry both a bacterial origin of replication and an antibiotic resistance gene for use as ...bacteria. Scientists have made many genetic modifications to create bacterial strains that can be more.... Pro-Tips Commercial competent cells range significantly in their transformation efficiency. The lowest...plasmid DNA for the purposes of storage and amplification. Higher efficiency cells are more important ...antibiotic containing agar plate. This step is not critical for Ampicillin resistance but is much more important...

...You may also like... Kit-Free DNA Purification Agarose Gel Purification Molecular Biology Reference Introduction...information on nucleic acid quantification, see our protocol for DNA quantification , which can be modified... not affect the quality of RNA or downstream applications. To improve yield of RNA, instead of incubating...the pellet. Air-dry the pellet for 5-10 minutes. Critical It is important to not let the pellet get too ...

... then repaired during the replication cycle. Empty vectors for LIC typically employ type II restriction... plasmid together through the transformation/replication process. LIC employs long overhangs to form a...polymerase can create overhangs of varying length (typically 10-12 bp) based on a specific sequence. The annealed...mixture by gel electrophoresis followed by gel purification . The cut vector end will now look like this...back the guanosine residue and become stalled. A typical T4 Pol reaction is shown. Mix the components (...μl Step 4: Amplify Insert by PCR Perform PCR amplification of your insert following the instructions provided...following step. This may be accomplished by gel purification . Step 5: Create Insert Overhangs Treat your...

...lab Over-Agar Antibiotic Plating Protocol AAV Purification Purify adeno-associated virus from a preparation...preparation produced in mammalian cells AAV Purification by Iodixanol Gradient Ultracentrifugation Protocol...culture Aseptic Technique Aseptic technique is a critical skill for success in numerous fields such as microbiology... AAV Titration by qPCR Use qPCR to measure the physical titer of your adeno-associated virus preparation...separating DNA by size for visualization and purification. Agarose Gel Electrophoresis Protocol Purifying...Purifying DNA from an Agarose Gel Gel purification allows you to isolate and purify DNA fragments based on... opens in a new window) Video Link Description Jessica Welch, PhD In this first installment of the Addgene...

... that you cut depends on your application. A diagnostic digest typically involves ∼500 ng of DNA, while...depending on application and is largely determined by the volume of DNA to be cut. A typical restriction...manufacturer’s instructions. Pro-Tip Depending on the application and the amount of DNA in the reaction, incubation...you will be using the digested DNA for another application (such as a digestion with another enzyme in a...70 °C for 15 mins, or purifying the DNA via a purification kit, such as a (Link opens in a new window) ...enzymes cut sequences which are similar, but not identical, to their recognition sites. This is due to "...