... 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 "...

...for short term studies of protein function. We specifically use this protocol with a subclone of HEK293T...perform your experiment Equipment Class II, Type A2 Biological Safety Cabinet 0.5–10 µL single channel pipette... or sodium hydroxide to adjust the pH to 7.0. Typically, the solution will be basic and will need adjustment...Before You Start The health of the cell line is critical for obtaining high levels of virus. HEK293T cells...optimal mass DNA:mass PEI ratio will need to be empirically determined for each new batch of 1 mg/mL PEI ...batches of PEI depending on the user, quantities of chemical used, volumes, pH adjustment etc. Consequently...inhibit transfection; therefore, plasmid DNA purification should include an endotoxin removal step. For...

...Electrophoresis DNA Ligation Background Information Gel purification allows you to isolate and purify DNA fragments...restriction enzyme -based cloning. Protocol: Gel Purification Follow the Agarose Gel Electrophoresis Protocol...Protocol with the following amendments: Notes: Gel purification is most efficient with lower % agarose gels,...available. Unlike the plastic tray, this will not significantly reduce the UV, but will protect the UV box from... This is especially important during the DNA purification step, as many kits cannot handle more than a... is most commonly done with a commercial gel purification kit, such as the (Link opens in a new window...purified DNA. Find more information about DNA quantification here . Tips and FAQ How do you get better resolution...

...lines of interest Equipment Class II, Type A2 Biological Safety Cabinet 0.5–10 µL single channel pipette...selective medium is used, it may have additional biological effects on the cell line. In the absence of selective... medium throughout experiments be determined empirically, and that the cell population be monitored regularly...orange/yellow if using a standard phenol red pH indicator) because the buildup of waste products could be...individual cells by passing several times through a serological pipet or by passing through a 40 µm cell strainer...expressing Cas9. A549 cells were transduced with lentiCas9-Blast 1 and then selected with 1 μg/mL blasticidin...lines generated from A549 cells transduced with lentiCas9-Blast 1 . A549 cells were transduced (MOI = 37...

...should have identical sequences on the ends (sequences A and B in the figures). These identical sequences...are significant amounts of undesired product, gel-purify DNA segments. Otherwise, PCR purification or ...PCR with primers that contain a 5′ end that is identical to an adjacent segment and a 3′ end that anneals...homology region. Hairpins in this region can significantly reduce the efficiency of two homologous ends..., Hutchison CA, Venter JC, Merryman C. (2010). Chemical synthesis of the mouse mitochondrial genome. Nature...

...resistant colonies Equipment Class II, Type A2 Biological Safety Cabinet 0.5–10 µL single channel pipette...tissue culture treated dish, Corning 3516 15 mL conical tubes, VWR 21008-216 Lentivirus preparation Reagent...You Start The health of the target cell line is critical for obtaining accurate titers. Check the cells...required to kill your target cell line needs to be empirically determined. Treat the target cells with a range... can usually be omitted because this dilution typically produces a lawn of cells, which cannot be accurately...Figure 1: A549 cells were transduced with the indicated serial dilutions of the lentiviral vector pRosetta...Figure 2: A549 cells were transduced with the indicated serial dilutions of the lentiviral vector pHAGE...

...stable cell lines Equipment Class II, Type A2 Biological Safety Cabinet 0.5–10 µL single channel pipette...tissue culture treated dish, Corning 3516 15 mL conical tubes, VWR 21008-216 Lentivirus preparation Appropriate...You Start The health of the target cell line is critical for obtaining accurate results. Check the cells...lower dilutions depending on your downstream applications. If you’ve titered your virus beforehand, you...and/or harvest to test for protein expression. Typically, cells transduced with lower dilutions of the ...expressing Cas9. A549 cells were transduced with lentiCas9-Blast and then selected with 1 µg/mL blasticidin...formed by expansion of single cells for 13 days. lentiCas9-Blast was a gift from Feng Zhang (Addgene plasmid...

...Immunocytochemistry Protocol Recombinant Antibody Purification Protocol Antibody Guide Introduction This protocol...primary antibody to use will vary and needs to be empirically determined. If possible, run an initial test ...Bovine serum albumin (BSA), VWR 10273-264 50 mL conical tubes, VWR 89039-656 96-well polyester (clear) ...antibody concentration will vary and needs to be empirically determined. We suggest starting with three concentrations... rpm for 15–30 min . Pro-Tip Check the plate periodically to determine when the desired color change has...display a green product. A precipitated product indicates the presence of too much HRP and the need to optimize...curve will vary between targets and needs to be empirically determined. The dilution series created in section...

...post on ddPCR for AAV quantification. This protocol was modified from the publication Lock et al. (2014) ...Adeno-associated viral vectors (AAV). This protocol specifically uses primers and probes targeting the ITR elements... is genome copies and generally represents the physical number of viral particles. Users may need to run...biosafety regulations. Equipment Class II, Type A2 Biological Safety Cabinet, Labconco 302411100 Droplet Digital...cartridge holder with bleach and place in the Biological Safety Cabinet (BSC). Make sure that the BSC ...Pro-Tip Prepare the viral dilutions in a separate biological safety cabinet (dilution BSC) than that used ...

...larger conical tubes. Depending on the centrifuge their speed ranges can vary, but are typically lower ...Tabletop Centrifuges sit on your benchtop and typically hold the common 1.5 mL microfuge tubes used in...volume as your samples. If your samples are significantly higher density than water, then you may need...according to speed and time (and temperature, if applicable) listed in your protocol. Pro-Tip Spin speed ...centrifuge begins to wobble or shake, this could indicate an unbalanced or improperly loaded centrifuge....

...) to titer lentivirus vectors. This protocol specifically uses primers and probes targeting integrated... sample. This protocol was modified from the publication Wang et al. (2018) . Before Starting Thaw the...ddPCR and analysis Equipment Class II, Type A2 Biological Safety Cabinet Aspirating unit Microcentrifuge...Scientific, 10199-452 Reagents GeneJet Genomic DNA Purification Kit, Thermo Fisher, K0721 6-well tissue culture...the next dilution. Repeat for all dilutions as indicated in the chart below. Dilution Factor Virus Volume...genomic DNA according to the GeneJet Genomic DNA Purification Kit instructions. Determine the concentration...cartridge holder with bleach and place it in the Biological Safety Cabinet (BSC). Make sure that the BSC ...

...pairs) for visualization and purification. Electrophoresis uses an electrical field to move the negatively...eye protection and gloves when working with this chemical. Note: If you add EtBr to your gel, you will also...approximately 75-80% of the way down the gel. A typical run time is about 1-1.5 hours, depending on the...instructions on how to do this, visit the Gel Purification page. Tips and FAQ How do you get better resolution...polyacrylamide gel electrophoresis (PAGE), which is typically used to separate 5 - 500 bp fragments. How do ...

...polymerase that can withstand radical temperature changes during a typical PCR. The DNA polymerase has ...template), a researcher can create thousands of identical copies using a simple set of reagents and a basic...GC-rich templates. What does each ingredient specifically do? Template DNA: Contains the portion of DNA...Buffer with MgCl 2 : Provides an optimal and stable chemical environment for the DNA polymerase to work adequately... provide a suitable environment for the DNA amplification reaction. Reference Page Top Index...

...after working in the lab. Ensure that a designated chemical waste accumulation site is present, and that you...before starting the work. Before working with chemicals, first review their material safety data sheets...waste that may be contaminated with pathogens or chemicals that present a danger to people and the environment...disposal, commonly done with an autoclave. Only mechanical pipetting should be done in the laboratory. Don... evacuate and close off the area and alert the chemical hygiene officer. BSL-2 Guidelines Remember, the...

...nucleotides together. After ligation, the insert DNA is physically attached to the backbone and the complete plasmid...digestion . Most restriction enzymes digest DNA asymmetrically across their recognition sequence, which results...troubleshooting failed ligations. The following table indicates the various controls: Control Ligase Interpretation... treated vector Insert or water + Any colonies indicate contamination of intact plasmid in ligation or... 3:1 ratio is not working or when doing more complicated cloning. While 3:1 will get you in the ballpark...

...medium. This dot is composed of millions of genetically identical bacteria that arose from a single bacterium...plate to inoculate a bacterial culture for DNA purification will minimize the chance of having a mixture...draw in discontinuous lines. Start by streaking a vertical line of bacteria along one edge of the plate. ...

...Diagnostic Restriction Digest Introduction Sequence verification of important plasmid features (such as the gene...recommend 5’ and 3’ sequencing primers. These primers typically anneal to the backbone and can help you verify...sequencing results as a trace file (.ab1) which graphically depicts the sequence as a series of colored peaks...unreliable. Although your sequencing results may indicate bases at specific locations, by looking at the...

... . The pattern of the fragments on the gel can indicate if the plasmid contains the expected size insert...before use of more expensive forms of plasmid verification, such as DNA sequencing . In the example above...or enzymes) that cuts within the insert, but significantly off center (ideally around 1/3 of the way from...side of the insert, you can get a very obvious verification of the orientation so long as the expected products...

... these cells can still be used in downstream applications. Safety Warnings Lentivirus is generally considered...fluorescent cells Equipment Class II, Type A2 Biological Safety Cabinet 0.5–10 µL single channel pipette...tissue culture treated dish, Corning 3516 15 mL conical tubes, VWR 21008-216 Lentivirus preparation Reagent...You Start The health of the target cell line is critical for obtaining accurate titers. Check the cells...

...mounting medium Laboratory wipes 15 mL conical tubes 50 mL conical tubes Before Starting Refer to the manufacturer's...Guide Western Blot Protocol Recombinant Antibody Purification Protocol Introduction Immunocytochemistry is...as methanol or acetone may be better for some applications. Remove the paraformaldehyde and follow your...

...dispensed. The boxes that the tips come in often indicate a volume range that the tip can hold. This should... using, you will be rotating dials behind the numerical display or the top of the plunger of the pipette...no liquid should drip from the tip. This could indicate that the tip is not on the pipette properly. Place...pipette tip by holding the pipette tip over your dedicated waste container and pressing on the tip ejector...