... Protocols Ligation Independent Cloning Ligation Independent Cloning You...for Ligation Independent Cloning Protocols...Molecular Biology Protocols Overview Ligation Independent Cloning (LIC) obviates the need for the time-...

...Validation Using the Indirect ELISA Method Antibody Validation Using the Indirect ELISA Method You may...This protocol describes how to run an indirect ELISA against a purified antigen to demonstrate the expected...Introduction This protocol describes how to run an indirect Enzyme-Linked ImmunoSorbent Assay (ELISA) against...display a green product. A precipitated product indicates the presence of too much HRP and the need to optimize...

...(Link opens in a new window) or isolating individual cells with cloning cylinders. Workflow Timeline This...when scanning the plate for individual cells, which can be difficult to find. Leave the cells undisturbed...medium (see below for more details) Day 1: Seed individual cells in a 96-well plate Day 2–14: Monitor cells...Some cell lines tend not to grow well in sparse/individual cultures due to lack of secreted growth factors...can be used to enhance the growth of cells in individual cultures. Other methods for enhancing single ...orange/yellow if using a standard phenol red pH indicator) because the buildup of waste products could be...trypsinization and break up any cell clumps into individual cells by passing several times through a serological...

...column at the indentation in the tip. Equilibrate the column with 10 mL of RT Protein A/G binding buffer. re...kDa MWCO column, Millipore Sigma UFC903024 50 mL LoBind tubes, VWR 76289-498 Before Starting Wipe down ...bleach prior to use. Warm the affinity column, binding buffer, and elution buffer to room temperature ... 7.0 Autoclave or filter sterilize Protein A/G binding buffer (20 mM sodium phosphate buffer, pH 7.0) ...protease inhibitor cocktail. Add 1 part Protein A/G binding buffer to 1 part tissue culture supernatant and...column. Pro-Tip Gravitrap columns have a finite binding capacity. If your sample exceeds the capacity, ...multiple columns. For information about the total binding capacity for your columns, refer to the manufacturer...

...chart, such as the (Link opens in a new window) double digest finder tool from NEB . In a 1.5mL tube combine...Link opens in a new window) NEB's compatible cohesive ends chart . If you cannot find compatible sticky ...restriction enzymes, visit (Link opens in a new window) NEB . Restriction enzyme digestion is commonly...amount of enzyme for your reaction. However, keep in mind that restriction enzyme activity is determined under...purification kit, such as a (Link opens in a new window) QIAGEN DNA cleanup kit . See the enzyme manufacturer's...lose their function. If you are having difficulty finding an enzyme that cuts your vector's multiple cloning...certain restriction sites. See (Link opens in a new window) NEB's table of methylation sensitive restriction...

...HiFi (Link opens in a new window) and Gibson Assembly (Link opens in a new window) . They also sell ET SSB... new window) . NEB has other resources, such as a primer design tool (Link opens in a new window) . Gibson...Link opens in a new window) OpenWetWare: Gibson Assembly (Link opens in a new window) OpenWetWare: Janet...Pro-Tip Add Extreme Thermostable Single-Stranded DNA-Binding protein (ET SSB) to the isothermal reaction mix... New England Biolabs (NEB) (Link opens in a new window) sells DNA Assembly kits and master mixes, including...doi.org/10.1038/nmeth.1318 (Link opens in a new window) Gibson DG, Smith HO, Hutchison CA, Venter JC, ...doi.org/10.1038/nmeth.1515 (Link opens in a new window) Rabe BA, Cepko C. (2020). A Simple Enhancement...

...Hybridization Sequence: The region of the primer that binds to the sequence to be amplified (usually 18-21bp... we want to amplify and design primers that will bind to and replicate it. The following image shows the...5'-ATGTGGCATATCTCGAAGTAC-3' for the region that binds the ORF and we will add the EcoRI restriction site...end of a linear piece (see (Link opens in a new window) NEB for more information). Thus, we recommend ...internet search will lead you to (Link opens in a new window) here and many others). If we put the sequence ...reaction using a kit, such as the (Link opens in a new window) QIAquick PCR Purification Kit . The PCR product...anticipated size and that your band is strong (indicating that the PCR reaction worked and that you have...

.... You can find even more video content on the Addgene Video Page . We hope that you'll find these protocols...cells Antibody Validation Using the Indirect ELISA Method Run an indirect ELISA against a purified antigen... Addgene Protocols On this page you'll find practical lab protocols that you can use for a wide...Lab Bench Name Description (Link opens in a new window) Link to Video Personal Protective Equipment (PPE...Molecular Biology Name Description (Link opens in a new window) Link to Video Making LB Agar Plates Create plates...Plasmid Cloning Name Description (Link opens in a new window) Link to Video Restriction Cloning Assemble plasmids... DNA fragments in a single reaction Ligation Independent Cloning (LIC) Scarless cloning with Type II restriction...

...ultracentrifugation. The arrow indicates the 60–40% interface. The vertical black line indicates the location of the... 973-985 (Link opens in a new window) . Right panel: cartoon indicating the position of the needle for...available under the name OptiPrep (Link opens in a new window) from Sigma-Aldrich. Protocol Video Watch the protocol...therapy 6.6 (1999): 973-985 (Link opens in a new window) . Figure 2: Left panel: Iodixanol gradient after...adeno-associated viral vectors," (Link opens in a new window) copyright (2006). Figure 3: Example of an SDS-... doi:10.1089/hgtb.2015.051 (Link opens in a new window) . Last reviewed on: November 7, 2023...

...sticky ends", are what allow the vector and insert to bind to each other. When the sticky ends are compatible...standard ligation reaction. Use a (Link opens in a new window) ligation calculator to easily quantify how much...take a fresh tube, thaw it one time and aliquot individual tubes of 5, 10 or 20μL for storage at -20°C. ...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...every available vector end. (Link opens in a new window) Ligation calculators are easily found on the web...exactly how much of each to use. Reference Page Top Index...

...a new window) Qiagen , (Link opens in a new window) Invitrogen , and (Link opens in a new window) Promega.... For details on how to streak a plate to get individual colonies and to generate liquid bacterial cultures...plasmid purification without using a kit, you can find a protocol for kit-free plasmid mini-prep at the... DNA or apply the solution to a column that will bind the now precipitated DNA. Wash the pellet or column...resuspension buffer of most kits. Reference Page Top Index...

...that the only version of full-length cDNA you can find for YGOI is in a bacterial expression vector. Using...are creating a fusion protein) Ideally, you will find two different restriction enzymes for your subcloning...cloned in the correct orientation. If you cannot find enzymes that meet these criteria, do not fear. You...restriction enzyme buffer (see (Link opens in a new window) New England Biolabs for more information about...from your transformation will give you the first indication as to whether your transformation worked. our...Finished Plasmid Finally, you will need to pick individual bacterial colonies and check them for successful... and can begin your studies. Reference Page Top Index...

...recombination can all impact the representation of individual plasmids in the pooled library during amplification...designed to be as general as possible but note that individual libraries may require modifications dictated ...satisfactory results for libraries up to 200,000 individual plasmids and can be scaled according to the actual...cells is sufficient for libraries up to 200,000 individual plasmids. For larger libraries scale the number...but this dramatically increases the chances of individual plasmids being lost from the pool and/or increasing...like Glycerol or DMSO solutions. Quantify the individual Maxipreps by Nanodrop, Picogreen, or Qubit. Each...or reagents are significantly overloaded. If you find that your pellet outstrips the capacity listed in...

... Test a variety of brands and FBS lot numbers to find one suitable with your protocols. FBS can be purchased...Check the cells for mycoplasma (Link opens in a new window) regularly Do not over or under-grow your cells...of resistant cells start proliferating and the individual wells become confluent, expand into larger vessels...Generation of monoclonal cell lines from expansion of individual A549 cells stably expressing Cas9. A549 cells...for 9 days. Single cells were then plated in individual wells of a 96-well plate and left undisturbed...Methods. 2014 Aug;11(8):783-4 (Link opens in a new window) . Last reviewed on: September 9, 2023...

...can hold 5-10 mL of agar and on which you can individually distinguish a maximum of ~100 colonies Pro-Tip...Tetracycline 10 mg/mL 10 µg/mL Notes: Unless otherwise indicated, the antibiotic powder can be dissolved in dH ..., you should prepare your plate pouring station: Find an empty section of lab bench with a working flame... looks like in your particular plate. For the remainder of the plates, pour directly from the bottle. ...test results. Sample Data In all cases below (-) indicates that the tested strain is not supposed to be resistant... resistant to the antibiotic, (+) indicates that the tested strain is supposed to be resistant to the ...

...transfection. Test a variety of brands and lots of FBS to find one suitable with your protocols. FBS can be purchased...Check the cells for mycoplasma (Link opens in a new window) regularly Do not over or under-grow your cells... the minimum concentration (Link opens in a new window) required to kill all of the cells. Use this dose...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...

...Videos Browse Addgene's collection of video content. Find video protocols, tips on using Addgene's resources... real Addgenies! Protocols (Link opens in a new window) Video Link Description Related Page Making LB ...Gel Protocol How-To Videos (Link opens in a new window) Video Link Description Related Page MTA Guide ...Depositing Page Career Videos (Link opens in a new window) Video Link Description Jessica Welch, PhD In this...

... The DNA polymerase is also stable enough to now bind to the primer DNA sequence. Extend DNA for 1 minute... to design primers . Note: (Link opens in a new window) Primer3 is an excellent resource for choosing ...Multiply the volume of each reagent by the number of individual PCRs you wish to perform and add 10% extra to... DNA amplification reaction. Reference Page Top Index...

... to protein (260/280) is generally used as an indicator of the purity of DNA samples. These days, many...1.80-2.00). Repeat for each sample. Notes: Keep in mind that despite the accuracy of the NanoDrop, if two... DNA in a buffered solution. Reference Page Top Index...

...Carbenicillin plated over-agar Plate shows several individual colonies and effective selection. 150 µL of 2...Carbenicillin plated over-agar Plate shows less individual colonies than the 1 mg/mL plate and effective...Carbenicillin plated over-agar Plate shows several individual colonies with smaller size than the 1 mg/mL and...