...buffer as the one in the gel box (do not mix different buffers and do not use water). Microwave for 1-3...instructions on how to do this, visit the Gel Purification page. Tips and FAQ How do you get better resolution...min until the agarose is completely dissolved (but do not overboil the solution, as some of the buffer ...to the running buffer when you run the gel. If you do not add EtBr to the gel and running buffer, you will...the DNA in your sample lanes. For more details on doing diagnostic digests and how to interpret them please...typically used to separate 5 - 500 bp fragments. How do you get better separation of bands? If you have similarly...

...how to design primers for cloning purposes. What do I do if my PCR isn't working? Try adding 1µl of 25mM...36.8 μL Sterile dH 2 O (variable) Note: If you are doing multiple PCRs, save time by creating a “master mix...added to the master mix. Forward and Reverse Primers DO NOT get added to a master mix. Place reaction tubes...reverse primers to the thinned walled PCR tubes. Note: Do this before adding the master mix so you know that...according to the length of the target sequence. FAQ How do I design primers? See our reference page on how to...The ideal setup for this troubleshooting step is to do one reaction with each, and one reaction with both...templates. What does each ingredient specifically do? Template DNA: Contains the portion of DNA that we...

...transformation . Tips and FAQ Do controls When doing ligations you should ALWAYS do a vector alone + ligase ...you know has only been thawed once before. Always do controls. See Tips and FAQ below for details. Try...situations where the 3:1 ratio is not working or when doing more complicated cloning. While 3:1 will get you...

...total plasmid DNA. Tips and Troubleshooting What do I do if my transformation efficiency is not high enough... Pooled Libraries Molecular Biology Reference How do I process my Addgene pooled library? Introduction...Endura from two separate transformations). Pro-Tip Do not pipette repeatedly or mix when removing SOC containing...Critical Be careful not to rip or shred the agar. Do so by gentle spreading. Some spreaders have a sharp...Maxi Kit (one conical is its own Maxiprep). Critical Do not freeze pellets for later purification. Immediately...colder reagents (not including the recovery media!). Do not proceed with Maxipreps or NGS until adequate ...representation is maintained. Maxipreps - Less is more: Do not overload the Maxipreps as yield can dramatically...

... given sequence. You want to choose enzymes that: Do not cut within your insert. Are in the desired location...(usually in the Multiple Cloning Site (MCS)), but do not cut elsewhere on the plasmid. Bonus: It is helpful...GAATTCATGTGGCATATCTCGAAGTAC-3'. Many restriction enzymes do not cut DNA efficiently at the end of a linear piece...any 6 bases, but you should ensure that the bases do not result in the formation of a hairpin structure...empty plasmid, you will still have colonies when you do not add ligase. If the colonies are a result of recipient...significantly more colonies when you add ligase. If you do not see any colonies, you should conduct a positive...

...want to choose enzymes that: Flank your insert, but do not cut within your insert Are in the desired location...(usually in the Multiple Cloning Site (MCS)), but do not cut elsewhere on the plasmid Will result in your...you cannot find enzymes that meet these criteria, do not fear. You have other options, such as: Adding...empty plasmid, you will still have colonies when you do not add ligase. If the colonies are a result of recipient...significantly more colonies when you add ligase. If you do not see any colonies, you should conduct a positive...

...little excess gel around the band as possible. To do so, it is often important to take the excised band... about DNA quantification here . Tips and FAQ How do you get better resolution of bands? A couple simple...gel comb; or c) loading less DNA in the well. How do you get better separation of bands? If you have similarly...

...strategy, sometimes it cannot be avoided. If you do have to do so, there is no way to control which orientation...vector backbone and therefore the only thing left to do is identify the clone(s) in which the insert is in...

...bacteria containing that plasmid from bacteria that do not contain it by artificial selection (i.e. growing...growth (bottom) Loosely close the cap on the bottle (do NOT close all the way or the bottle may explode!)...concentration ( see table below ). Note: If you intend to do a mini-prep you will usually want to start 2 mL in...

...wear it the whole time you are working in the lab. Do not eat, drink, chew gum, or apply makeup in the ...way. It is the easiest way to ensure that you’re doing your work in the safest way possible. Protecting...this SOP in another location, please note that you do so at your own risk; you should ensure that any local...

...filter or centrifugation at >500 x g for 5 minutes. Do not use the medium if the cells are overly confluent... solution into each well of a 96-well plate. By doing this, you are seeding the plate at an average density...After ~7 days, scan the plate for cell growth. If you do not observe any colonies, continue incubating the... one colony. Wells with more than a single colony do not contain a monoclonal population and should be...expansions (monoclonal, 1-5). Wild-type A549 cells do not express Cas9 and are included as a control (WT...

...regularly Do not over or under-grow your cells. Thaw a new vial of cells after 20-30 passages. Do not add...determine the optimal dose of selective reagent for your target cell line. To do this, treat target cells...surviving cells in the culture, so it is important to do regular media changes and maintain optimal growth...cells with a range of doses of antibiotic and determine the lowest dose that kills all of the cells. Prepare...should be changed every 2–3 days to maintain the dose of antibiotic, which may not be stable at 37 °C....

...way. It is the easiest way to ensure that you’re doing your work in the safest way possible. Protecting...this SOP in another location, please note that you do so at your own risk; you should ensure that any local...

...Gently mix sample (do not vortex) Incubate 30 min at 37 °C Transfer to ice ** Critical: do NOT treat your ...plates) and store at -20 °C. Once a standard is thawed do not freeze it again but store at 4 °C and use within... duplicate Load 5 μL of each sample in duplicate. Do not forget to include a no template control ( NTC...

...broad stroke. Only touch the surface of the plate, do NOT dig into the agar. Another very popular technique...bacterium. If the bacterial growth is too dense and you do not see single colonies, re-streak onto a new agar...

...tags, mutations and a portion of the insert, but we do not sequence the entire plasmid. Addgene strongly... match Addgene’s sequencing result, what should I do? Check your trace file first; the apparent mismatch...

... disperse mixture evenly. Do not pipette or swirl too vigorously, as you do not want to dislodge the cells...antisense sequences from step B.1 into the oligos below. Do not change the ends; these bases are important for...12-15 hours. c. In polypropylene microfuge tubes (do NOT use polystyrene tubes), make a cocktail for each...first. Pipette FuGENE® directly into the OPTI-MEM – do not allow FuGENE® to come in contact with the walls...media through a 0.45 μm filter to remove the cells. Do not use a 0.2 μm filter, as this is likely to shear...virus-containing media and replace with fresh media. Do not add puromycin until at least 24 hours after infection...

...yield. Do not overgrow your cells. Pass the cells twice a week during the maintenance phase and do not allow...Rapid-Flow PES Filtration Unit, Nalgene 167-0045) Pro-Tip Do not use filters made of materials other than PES..... AAV particles stick to many other surfaces, but do not stick to PES. Using a PES filter will maximize...

...bacteria containing that plasmid from bacteria that do not contain it by artificial selection (i.e. growing... of the bottle with its cap or aluminum foil (but do not make an air-tight seal!) and tape the bottle ...molten gel-mix in the water bath for at least 5 min. Do not let any of the water bath water touch the neck...

...regularly Do not over- or under-grow your cells. Thaw a new vial of cells after 20–30 passages. Do not add...