...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...

...near optimal wavelengths (Fluc emits at 610 nm) and do not require external light sources. Unfortunately...

...systems listed above are orthogonal, meaning that they do not affect each other. For example, the GAL4 transcription...

... 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...

...you’ve introduced your RNAi into your organism, how do you identify the actively silenced ones? Here, I’...

...success, we find it is usually faster and easier to do the validation first.  AAV-mediated gene expression...

...recombinant clones are generated.  A simple way to do this is to express lambda red genes from a plasmid...

...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...

...transforming large plasmids (>10 kb) or BACs, what can I do? Chemically competent cells are fast and easy to ...cell/DNA mixture to induce membrane permeability. To do this you will need to have access to an electroporator...

...excitation or optogenetic inhibition. First things first: do you want to turn ON or turn OFF neurons in your experiment...Examples: iChloC, SwiChRca, Phobos, Aurora Browse Channelrhodospin plasmids . Halorhodopsins Halorhodopsins are...

...μm) tissue sections and 3D cultures These samples do not necessarily require optical sectioning, but open...while also avoiding the toxic effects of high light doses to the cells. Thin static samples Ex: Fixed monolayers...out of focus light. However, they also continually dose the sample from top to bottom with excitation light...

...to G (or T to C) change. Adenosine DNA deaminases do not exist in nature, but have been created by directed...have been developed for inhibition using dCas9. How Do You Use a CRISPR Library? CRISPR libraries from Addgene...alterations, such as small mutations or inversions, than do large deletions generated by Cas9 systems. Cas3 must..., Cogan, J. Z., Replogle, J. M., Adriaens, C., Ramadoss, G. N., Shi, Q., Hung, K. L., Samelson, A. J.,...

...box and push the end of the pipette onto the tip. Do not touch the tips with your fingers to avoid contaminating...plunger, gently lower the pipette tip into the liquid. Do not submerge the pipette itself into the liquid and...

...regularly Do not over or under-grow your cells. Thaw a new vial of cells after 20–30 passages. Do not add...Procedure Before beginning a colony formation assay, the dose of antibiotic required to kill your target cell ...determined. Treat the target cells with a range of doses of antibiotic. Determine the minimum concentration...window) required to kill all of the cells. Use this dose for the colony formation assay. Prepare a batch ...

...thicker as the proteins and DNA are denatured. Pro-Tip Do not vortex at this stage or the genomic DNA will ...4. Note: Phenol-chloroform is a hazardous waste - DO NOT pour down sink. Top Protocol: Ethanol Precipitation...

...Media 10 mL 10% Pluronic-F68 40 mL 200 mM Glutagro Do not add selective reagents Store at 4 °C until use...incubator on a shaking platform set to 120 rpm. Pro-Tip Do not use cells that are over 30 passages. Section ...

...complementary to template strand). However, primers do not need to correspond to the template strand completely...