...spectrophotometer. A spectrophotometer uses the absorbance/transmission of light through a liquid to determine the concentration...measured under the 260/280 column (A good purity ranges from 1.80-2.00). Repeat for each sample. Notes:...

...Digest of Plasmid DNA DNA Ligation Bacterial Transformation Summary Oligo overlap cloning can be used anytime...the vector with 0.75-6 ng of annealed oligos). Transform 2-3μL into your favorite competent bacteria and...

...your cell culture dish. Microscopes come in a huge range of shapes and sizes - from phone-sized, foldable...powerful (but are cheap and accessible) to massive transmission electron microscopes that allow us to see cellular...

...the solution has a tendancy to boil over. Placing saran wrap over the top of the flask can help with this...are usually referred to as ‘bands’ due to their appearance on the gel. Pro-Tip If you will be purifying ...

...x (0.220 L) = 8.14 g pre-mixed LB-agar powder. Transfer the LB-agar powder you’ve measured out into an...molten agar from boiling over in the autoclave. Transfer the sterile water (in our case 220 mL) to the ...

..., 1163–1171. PMID: 23979020 Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P. D...38688943 Hsu, P. D., Scott, D. A., Weinstein, J. A., Ran, F. A., Konermann, S., Agarwala, V., Li, Y., Fine..., 17 (5), 471–479. PMID: 32203383 Nishimasu, H., Ran, F. A., Hsu, P. D., Konermann, S., Shehata, S. I....CRISPR-Cas9. Cell , 167 (7), 1829-1838.e9. PMID: 27984730 Ran, F. A., Hsu, P. D., Wright, J., Agarwala, V., Scott...Nature Protocols , 8 (11), 2281–2308. PMID: 24157548 Ran, F. A., Cong, L., Yan, W. X., Scott, D. A., Gootenberg...may be a single-stranded oligonucleotide, double-stranded linear DNA, or a double-stranded DNA plasmid. ...including transposons, integrases, and recombinases, with Cas enzymes. CRISPR Transposases Transposon systems...

... can be used in transient transfections to achieve expression of the transgene, and those that can are.... Lentiviral transfer plasmids are not designed specifically for transient transfections. Therefore, there... spliced transcripts to facilitate nuclear export. Tat in trans Second generation only; Trans-activator...follows: Lentiviral transfer plasmid encoding your insert of interest. The transgene sequence is flanked...into the host genome upon viral transduction. Many lentiviral transfer plasmids are based on the HIV-1...lentiviral transfer plasmids must be used with a 2nd generation packaging system because transgene expression...heterologous promoter on the transfer plasmid. Expression of the transgene from this promoter is no longer...

...please see this blog post. Browse the article from Ran et al. , 2015 from the Zhang Lab to find plasmids...synthesis of the complementary DNA strand. Rep and Cap are translated to produce multiple distinct proteins...proteins). When constructing an AAV transfer plasmid, the transgene is placed between the two ITRs, and... replication. The transfer plasmid, Rep/Cap, and the helper plasmid are transfected into HEK293 cells,...typically processed into a double-stranded circular episome through double stranded synthesis. These episomes ...machinery to synthesize the complementary strand, transgene expression may be delayed. To overcome this... capacity of AAV, a longer transgene may be split between two AAV transfer plasmids, the first with a ...

... sense or coding strand of the transcribed gene. The coding strand is the DNA strand that encodes codons...to the mRNA transcript produced. The antisense strand is referred to as the template strand or non-coding...non-coding strand as this is the strand that is transcribed by the RNA polymerase. DNA sequences called response...initiation of transcription whereas transcription factors promote the initiation of transcription. The most...template for the translation of a protein. RNA polymerase III transcribes genes encoding transfer RNAs (tRNA...reference material covering promoters, transcription and the transcription complex. Plasmid... to DNA. RNA polymerase transcribes DNA to mRNA which is ultimately translated into a functional protein...

...γ-Retroviral transfer plasmid encoding a transgene, sgRNA, or shRNA of interest: The transgene sequence is...packaging and transduction are not encoded by the transfer plasmid, but instead are provided in trans by other...Pol in trans Precursor protein containing Reverse Transcriptase and Integrase components. Transfer cPPT ...integrated into the host genome upon viral transduction. Most transfer plasmids available from Addgene were ...packaging, and transfer) are supplied by three types of plasmids, which are cotransfected into the 293T...cell line. γ-retroviruses can transduce target cells, but the transduced target cell does not produce ...Type Element On Same Plasmid as Transgene? Purpose Envelope VSVG in trans Vesicular somatitis virus G glycoprotein...

...36-Kb double-stranded DNA (dsDNA) molecule containing multiple, heavily spliced transcripts. At either ...recombination of the transgene into the adenoviral plasmid. One can also co-transform standard BJ5183 with...pAdTrack Class of transfer vectors that contain IRES-GFP pShuttle Class of transfer vectors that do not...are divided into early (E1-4) and late (L1-5) transcripts. There are 57 accepted human adenovirus types...Deletion of these two components results in a transgene packaging capacity of >8 Kb. Constructs contain...to facilitate homologous recombination of the transgene into the adenoviral plasmid. Please see our Biosafety...consists of two types of plasmids: shuttle (or transfer) vectors and adenoviral vectors. Find vectors ...

...molecular biology, that proteins are translated from RNA, which is transcribed from DNA. DNA is comprised of ...Region Drives transcription of the insert. The promoter is designed to recruit transcriptional machinery ... a base on one strand pairs with a particular base on the other, complementary, strand. Specifically, ... single stranded DNA adding nucleotides according to the sequence of the complementary strand. The replication...on the random incorporation of modified, fluorescently tagged bases onto the growing DNA strand in addition...respectively) that are organized into a double stranded helix. The order of these 4 nucleotides makes ... determinant. Plasmids are fragments of double-stranded DNA that typically carry genes and can replicate...

...also like... Making LB Agar Plates Bacterial Transformation Recovering Plasmid DNA from Bacterial Culture...

...wavelengths, ranging from blue to yellow to red. Red light exhibits better tissue penetrance, which may ...including protein activation, membrane localization, and transcriptional activation. In the widely used...synthetic two-part transcription factors - Cry2 and CIB1 are fused to a transcriptional activation domain...trafficking signals to localize opsins to the cell membrane. Microbial opsins, such as those described below... at physiological pH and trafficks well to the membrane. Excitatory (Depolarizing) ChR Variants. The optogenetic...from Kir2.1 and ER export signal provide improved membrane targeting 589 Archaerhodopsins Arch Light-activated...neuronal activation/inactivation periods. These can range from msec (hChR2) to “long lasting,” e.g. seconds...

...For easy-to-transfect cell types (e.g. HEK293 cells), transfection with standard transfection reagents may...mammalian cell line that can be transfected at high efficiency Lentiviral transduction Cas9 and gRNA can be present...present in a single lentiviral transfer vector or separate transfer vectors May contain reporter gene ...screens using CRISPR AAV transduction CRISPR elements are inserted into an AAV transfer vector and used to ...ribonucleoprotein (RNP) complexes are advantageous due to the transient Cas9 expression. The table below summarizes the...details, see Plasmids 101: Mammalian Vectors ) Transient or stable expression of Cas9 and/or gRNA in a ... mammalian cell lines Useful for difficult to transfect cell types, can also be used in vivo A common ...

... gels, so you will want to stay in the 0.7-0.8% range if possible. You will want nice crisp bands. This...

...proteins by size. Transfer proteins from the gel to a membrane. Incubate the membrane with an unconjugated...block the membrane to remove excess antibody and prevent unwanted binding. Incubate the membrane with a ...Wash and block the membrane. Activate the HRP with a substrate kit. Image the membrane using either X-ray...hybridoma cells into plasmids. Plasmids are then transfected into cells and induced to express antibodies....variable regions, shown in light blue and light orange, that bind to a specific antigen, triggering an...constant region, and is shown in dark blue and dark orange. The constant region is a stable part of the antibody...responsible for the variable region goes through a randomized reorganization process, creating a unique binding...

... bacterial endonucleases that recognize a large range of DNA sequences. Given the variety of these enzymes...

... tubes. Depending on the centrifuge their speed ranges can vary, but are typically lower than some other...

...by certain bacteria and archaea, cleave double stranded DNA (dsDNA) at specific sequence sites in the ...into fragments containing precise 5' or 3' single-strand overhangs (sticky ends), or no overhang (blunt .... They also cut specific target sequences, that range from 4 to 13 base pairs, and produce predictable...' end of dsDNA fragments to generate 3' single-stranded overhangs. DNA polymerases synthesize DNA molecules... 4 nucleotides and lastly DNA ligase fuses DNA strands together. In Gibson assembly DNA fragments with...nicks in the DNA to create one piece of double stranded DNA. A major benefit of Gibson cloning is that...enzyme or Gateway recombination sites). Any double stranded DNA fragments can be used, so if properly designed...