...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....

...we can tell, DnaA doesn’t need to hydrolyze ATP to do its job — although ATP does get hydrolyzed over time...

...machinery such as Rep or other initiator proteins, they do not require host proteins for replication and can...spp., Rhizobium spp., Rhodopseudomonas spp., Rhodospirillum spp., Shewanella spp., Thiobacillus spp., Xanthomonas...

...biorender.com.   Of course, this is quite easy to do on a plot generated in Biorender! While a helpful...

...camera’s image to my remote location, live. After doing a bit of looking, I found an inexpensive HDMI transmitter...

...less stable than viruses produced in cell lines that do not express these sugars. (Takeuchi et al., 1997)...

...science.1138389 Le TTH, Tran NT, Dao TML, Nguyen DD, Do HD, Ha TL, Kühn R, Nguyen TL, Rajewsky K, Chu VT ...off-target editing, and, for RNA editing, fine-tuning dosage so that enough wild-type MECP2 is made inside the...

...your target base in the protospacer sequence. If you do not find a suitable ‘NGG’ PAM at your target genomic...

... extension can usually ensure that the 3’-base(s) do not form secondary structures. The introduction (...

...clinical grade hiPSCs, reprogramming methods that do not involve modification of the original genome by...

...find a positive one. So if you have better things to do than clone picking, choose your guide RNAs wisely...

...saturation mutagenesis with the same protocol. What do you think about one pot saturation mutagenesis? How...

...Genome-Wide Screening What Are Pooled Libraries? How Do You Use a Library? Cas9 Fusion Tools Activation/Repression...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.,...

...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...

...caption courtesy of Challis et al., 2022. Where do they come from? Systemic capsids are typically derived...efficacy can vary depending on species. LY6A and LY6C1 do not have known homologs in NHPs, and capsids that...mouse strains). AAV-PHP.eC, AAV9-X1.1 and AAV1-X1 do not display strain-specific tropism and can be used...2023). High viral titer High vector genome (vg) doses are recommended for all systemic capsid applications... AAV-PHP.eB is typically administered at a total dose between 1 x 1011 and 5 x 1011 vg per animal (i.e...mL titer), while AAV-PHP.S is typically used at a dose between 3 x 1011 and 1 x 1012 vg*. Most systemic...capsids are easy to produce in high titer, but higher doses (>1 x 1012 vg total in mice) may increase the chances...

...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...

...processes in different cell types. One great way to do this is to use CRY2-CIB1 clustering. Fuse either ...provided CIB constructs for targeting Golgi, ER, and endosome membranes) (Figure 4). The authors used SuperPLD...

..., phosphomimetic mutations on the activation loop do not fully activate MEK1. In fact, replacing just ...