...important to use a high fidelity taq polymerase to minimize mutations. The fidelity of the polymerase becomes...we recommend that you use a wide gel comb, run the gel on the slower side, and skip lanes between samples... Addgene’s Sequence Analyzer , to allow you to identify which restriction sites are present in a given... chance for mis-priming, so you can use a pretty wide range of annealing temperatures, but you may need...addition to a DNA ladder standard, it is also a good idea to run an uncut sample of each vector to help with...total DNA in a standard ligation reaction. You ideally want a recipient plasmid to insert ratio of approximately...

...should have identical sequences on the ends (sequences A and B in the figures). These identical sequences... each other. Phusion High-Fidelity DNA Polymerase - incorporates nucleotides to “fill in” the gaps in ...may also like... Cloning Methods Empty Backbone Guide Molecular Biology Protocols Summary In 2009 Dr. ...PCR with primers that contain a 5′ end that is identical to an adjacent segment and a 3′ end that anneals...opens in a new window) OpenWetWare: Janet Matsen’s guide to Gibson Assembly (Link opens in a new window) ...

...the microscope slide. Gently place the coverslip on the microscope slide with the cell side facing down....Plasmid Collection Antibody Blog Posts Antibody Guide Western Blot Protocol Recombinant Antibody Purification...antibody Secondary antibody Deionized water Microscope slide Anti-fade mounting medium Laboratory wipes 15 mL...and follow your institution's laboratory safety guidelines for disposing of waste in the appropriate container...instructions before starting your experiment and consider titrating your antibody to determine the optimal...

...Amplification You may also like... Pooled libraries Guide to Using Pooled Libraries All CRISPR Pooled Libraries... plasmids differ by only a short region of DNA: guide RNAs targeting different genes in an organism's ...applications. Repeated amplifications should be avoided as best as possible due to the inherent possibility... abrasively at a certain angle. Incubate plates upside down at 30 ℃ overnight. Critical Ensure at this...media drips onto the lid. Let plates remain agar side up until dried before overnight incubation if needed... number of colonies can be too great to count. Ideally, dilutions would have been sufficient to enumerate...beforehand! If you’ve already gone too far, weigh an identical empty tube. It will be close enough for the purpose...

...Protocol Video Before you begin this protocol, watch the protocol video below to get a quick idea of how...extract 10.0 g peptone from casein 10.0 g sodium chloride 12.0 g agar-agar 1 L Sterile H 2 O Sterile plates...and the medium they will contain including the identity of the antibiotic. Pro-Tip We batch label our ...particular antibiotics. Position the flame just to the side of where you’ll be pouring your plates - be sure... with a pipete for the first plate to get a good idea of what that volume looks like in your particular...

...Titration by qPCR AAV Viral Service Viral Vector Guides Virus Blog Posts Molecular Biology Reference Introduction... 10% bleach. Safety Warnings AAV is generally considered biosafety level 1 but may require BSL-2 handling...plate onto a chilled 96-well freezer block and set aside in the droplet generation BSC to cool. Prepare the...bottom of the tube without collecting drops along the side of the tube. Cap gently - do not push the cap in...bottom of the well. Lift the tips ~1 mm. Touch the side of the well and tilt the pipette tips at a 45° angle...slowly and gently dispensing the droplets down the side of the tube. Place a Pierceable Foil Heat Seal on... should be close to zero (B08). At Addgene, we consider runs with an NTC > 10 to be invalid. To reduce...

...check the identity of a plasmid by diagnostic digest . Last Upload: Oct. 11th, 2016 Protocol Video Watch ...restriction enzymes, allowing scientists to target a wide variety of recognition sequences. For a list of ...Watch the video below to learn how to analyze your restriction digest results. Equipment Electrophoresis chamber...restriction enzyme activity is determined under ideal conditions with very clean DNA, so using a little...enzymes cut sequences which are similar, but not identical, to their recognition sites. This is due to "...

...support reproducibility in science. General Considerations For antibody-expressing plasmids containing...antigen (EBNA). Safety Warnings HEK293 cells are considered biosafety level 2. Please ensure that you are...tubes Microcentrifuge tubes Cell counting chamber slide 30 mL luer-lock syringe, BD BD302832 0.2 µm luer-lock...salt, Sigma Aldrich P4543-10G Polyethylenimine hydrochloride, M.W. 40000 (PEI-MAX), Linear, Transfection ...BalanCD HEK293 Feed, Irvine Scientific 91166-500ML Benzamide, Millipore Sigma 12072 Antipain, Millipore Sigma...several hours. Adjust to pH 7.0 with 10 N sodium hydroxide (NaOH) or 5 N hydrochloric acid (HCl). Note: Adjust...

...we recommend that you use a wide gel comb, run the gel on the slower side, and skip lanes between samples...including Addgene’s Sequence Analyzer , allow you to identify which restriction sites are present in a given...plasmid (if you are creating a fusion protein) Ideally, you will find two different restriction enzymes...addition to a DNA ladder standard, it is also a good idea to run an uncut sample of each plasmid to help with...total DNA in a standard ligation reaction. You ideally want a recipient plasmid to insert ratio of approximately...

...sample is separated by denaturing polyacrylamide gel electrophoresis alongside serial dilutions of a standard...gel Day 1 or later: Image analysis Video Watch this instructional video to learn how to determine purity...Plasmid Collection Antibody Blog Posts Antibody Guide Transfection for Recombinant Antibodies Recombinant...in the cell (e.g., failure to cleave the signal peptide) and may not be functional. This sample would fail...

...nice crisp bands. This can be achieved by using a wider gel comb and running the gel at a lower voltage....the UV exposure of the DNA. Therefore, it is a bad idea to use a gel imager to take a picture of the gel... gel. Notes: To protect the UV box, it is a good idea to place the gel on a glass plate if available. ...down on the UV box and trim the top, bottom and sides with the razor blade. This is especially important...voltage for a longer period of time; b) using a wider gel comb; or c) loading less DNA in the well. How...

...for Lentivirus You may also like... Viral Vector Guides Virus Blog Posts Mol Bio Protocols Viral Service...applications. Safety Warnings Lentivirus is generally considered biosafety level 2+. Please ensure that you are... in the lab by heating to 56 °C for 30 min. Considerations Before You Start The health of the target cell...anticipate that your viral stock will be higher titer, consider additional dilutions. Dilution Volume of Lentivirus...cells in each well. When calculating titer, only consider wells with less than 40% fluorescent cells. Titering...

... Transfection You may also like... Viral Vector Guides Virus Blog Posts Mol Bio Protocols Viral Service...dish, Corning 430167 Hydrochloric acid Sodium hydroxide 0.22 μm polyethersulfone (PES) filter Syringes... the solution Use hydrochloric acid or sodium hydroxide to adjust the pH to 7.0. Typically, the solution...discard the tube and thaw a new working stock. Considerations Before You Start The health of the cell line...of pRosetta :µg of PEI. The 1:2 and 1:3 ratios provided high transfection efficiencies as can be seen ...

...Last Update: Nov. 13, 2017 Protocol Video Watch the protocol video below to learn how to isolate single...C water bath for 30-60 secs (45 secs is usually ideal, but this varies depending on the competent cells...outgrowth, for the other antibiotics it is a good idea to outgrow for at least 20-30 mins) I get very few...you need to transform large plasmids, it is a good idea to use electro-competent cells. Instead of relying...

...Lentivirus Production You may also like... Viral Vector Guides Mol Bio Protocols Introduction This protocol can...tubes, VWR 21008-216 Hydrochloric acid Sodium hydroxide 0.22 μm polyethersulfone (PES) filter Transfection...the solution. Use hydrochloric acid or sodium hydroxide to adjust the pH to 7.0. Typically, the solution...batch of 1 mg/mL PEI and for each cell line. Considerations Before You Start The health of the packaging...and 1:6. The 1:2 and 1:3 total DNA:PEI μg ratios provided high transfection efficiencies as measured by ...

...window) from Sigma-Aldrich. Protocol Video Watch the protocol video below to learn setup and use an iodixanol...Ultracentrifugation You may also like... Viral Vector Guides Virus Blog Posts Mol Bio Protocols Introduction...can collect fewer fractions once you have a good idea when the virus elutes from the gradients. In a biosafety...tube. (Optional) For first time users, it is a good idea to assay each fraction by silver stain or SYPRO ...

...with Lentivirus You may also like... Viral Vector Guides Virus Blog Posts Mol Bio Protocols Viral Service... in the lab by heating to 56 °C for 30 min. Considerations Before You Start The health of the target cell...the virus will have higher levels of expression. Consider expanding populations transduced with a variety...expressors may take over the culture. To overcome this, consider generating monoclonal lines from the early polyclonal... and is described in Improved vectors and genome-wide libraries for CRISPR screening. Sanjana NE, Shalem...

...Last Update: Feb. 28, 2017 Protocol Video Watch the protocol video below to learn how to isolate single...the plasmid name and the date. It is also a good idea to add the antibiotic resistance and your initials...This dot is composed of millions of genetically identical bacteria that arose from a single bacterium. If...

...Last Update: Oct. 27, 2017 Protocol Video Watch the protocol video below to learn how to spread antibiotic...Several other devices may be used for this purpose, provided that they fit your petri plate. Incubate the plate...

...phosphodiester linkages, which permanently join the nucleotides together. After ligation, the insert DNA is physically...the ligation process. If the sticky ends on either side of the vector are compatible with each other, the...which degrades upon freeze/thaw cycles, it is a good idea to take a fresh tube, thaw it one time and aliquot...