...glutamine such as glutaGRO, Corning 25-015-CI) Heat-inactivated FBS 1X PBS pH 7.4 without calcium or magnesium...bottle of DMEM high glucose, add 55 mL of heat-inactivated FBS and 5 mL of 100X glutaGRO. Store at 4 °C....performed at Addgene on the A549 cells described in our sample data , each 96-well plate gave rise to 15–20 monoclonal...solution will be used to seed the 96-well plate. Sample Calculation First, use a hemocytometer to quantitate...plate for individual cells, which should be on the same focal plane. Scan the entire plate and make note...highest or lowest transgene expression ( Figure 2 ). Sample Data Figure 1: Generation of monoclonal cell lines...

...spatiotemporal control of transgene expression or activity can be achieved using switches, including riboswitches...function like your average AAVs — and have many of the same restrictions, like limited cargo space (~4.7 kb)...systems, which use two or more AAVs to transduce the same cell, can accommodate larger, modular genetic payloads...efficient AAV-PHP.eB, developed and deposited by the same lab. AAV-PHP.eB is the most commonly requested systemic...off-target organs. AAV.CAP-B22 was developed through the same process as AAV.CAP-B10 and shows high transduction...

... the sample to that of a standard curve to extrapolate the concentration of antigen in the sample. A variety...with samples, eliminating them from the procedure removes one potential source of cross-reactivity.   While...use the same secondary antibody for any ELISA protocol that uses primary antibodies of the same isotype...-containing sample with their conjugated primary antibody. Users next incubate the sample with the multiwell...used to quantify the level of target antigen in a sample. While Engvall et al. originally developed the ... proteins and small molecules from a variety of sample types (Engvall 1971). Scientists value ELISAs for...using the method. ELISA basics All ELISAs follow the same overall process. First users coat a multiwell plate...

...the provirus). Contains sequences necessary for activation of viral genomic RNA transcription. R; Repeat...While both lentiviruses and γ-retroviruses use the same gene products for packaging (i.e., Gag, Pol, and... only in lentivirus. Are γ-retroviruses self-inactivating (SIN)? They can be. Plasmids that encode SIN.... Return to Top Glossary Plasmid Type Element On Same Plasmid as Transgene? Purpose Envelope VSVG in trans...

...presence of several biomarkers together in the same tissue sample predicts a certain cancer and want to look...you will need to start with a fixed sample. Depending on the sample type, this might be frozen or formalin-fixed...you image the first target, you inactivate the fluorophores in the sample, either by chemical treatment ...al, 2022 for a sample protocol.)  Simultaneous IF Depending on your needs, your sample, and targets, you...known to be abundant in your samples (with a primary antibody that has the same species and isotype as the...set of samples where you label only one target in each. Compare these individually-labeled samples with ...the sample is being damaged by all the handling steps, or there’s some unexpected cross-reactivity. Keep...

...two different shRNAs that target the same gene can produce the same phenotype will alleviate concerns about...plasmid in mammalian cells. sin 3’LTR 3’ Self-inactivating long terminal repeat. f1 ori f1 bacterial origin...boiling water. If using a PCR machine, incubate the sample at 70°C for 10 minutes then slowly cool to room...Biotechnol . 1997. 15(9):871-5. PubMed . Self-inactivating lentivirus vector for safe and efficient in ...

...than generally reactive ones. And make sure to check for any reported cross-reactivity when planning your...minimize the risk of cross-reactivity. To further limit species cross-reactivity, try to find secondary antibodies...cross-reactivity. For example, if you are using mouse IgG2a and rat IgG1 antibodies in the same application...specific secondary antibody, rather than a broadly  reactive secondary (Fig. 1). For example, an anti-mouse...Manning 2012).      Figure 1: Broadly reactive versus subclass-specific antibodies. Only a fraction...of the antibody molecules present in a broadly reactive secondary antibody will bind to a monoclonal primary...different isotypes and subclasses, use a broadly reactive secondary antibody to increase the chance of primary...

... or hydroxyurea, is necessary for lytic cycle activation. When recombinant AAV (rAAV) is used for research...toolkit for human gene therapy. Wu Z, Asokan A, Samulski RJ. Mol. Ther. 2006 Sep;14(3):316-27. Epub 2006...independently of DNA synthesis. McCarty DM, Monahan PE, Samulski RJ. Gene Ther. 2001 Aug;8(16):1248-54. PubMed ...

... replicates are repeated measurements of the same sample and are included to assess variability in the... the tau western blot several times with the same samples and compare the results across runs. If the ... wild-type and knockout samples. In this test, wild-type and knockout samples are treated with the identical...present in the wild-type samples and absent in the knockout.  If knockout samples are unavailable, try substituting...control, and cross-reactivity control when multiplexing. A, wild-type and knockout samples are the gold standard...interactions between the sample and the secondary antibody. C, a label control treats the sample to the full protocol...interpretation of results. A positive control is generally a sample or group that will have a desired response. For...

...recognize the same region, or epitope, of the target protein. These antibodies are also all the same isotype...heterogeneous mixture of many antibodies that recognize the same protein. In the immune system, antibodies are produced...peptide sequence derived from this protein. At the same time, an adjuvant such as KLH or Freund’s adjuvant... without causing high background due to cross-reactivity. When to use polyclonal antibodies One advantage...clones will still recognize your protein. For the same reason, they’re also useful in experiments where...experiment, you should either order several vials of the same lot of polyclonal antibody, or select a monoclonal...your polyclonal antibody, you’ll want to use the same positive control across experiments to be aware ...

... are the same isotype and therefore cannot be indirectly probed simultaneously on the sample. What to ...because the pair belong to the same isotype and will react with the same secondary. If you were to change...the indirect method since a secondary antibody reactive to a certain isotype will bind to all members ...that group. If you use two antibodies that are the same isotype then the secondary antibody will bind to... Figure 2: Two primary antibodies with the same isotype can not be used for multiplexed indirect...parental species which can be bound by broadly reactive secondary antibodies to that parental species....

...must selectively block and unblock repeatedly the reactive groups on a nucleotide when adding a nucleotide...primer is used to amplify a eukaryotic genomic DNA sample. However, a primer should not be too long (> 30...

...AATACCACTACAATGGAT (BD Biosciences) 3' end of Gal4 activation domain, forward primer GFP-F GGTCCTTCTTGAGTTTGTAAC...CCCTTGAACCTCCTCGTTCGACC (MSCV) Murine stem cell virus, same as MSCV, forward primer pMRB101-F AAGATGCAGGCAGCTGAGTT...GATTTAATCTGTATCAGG (Invitrogen) 3' of MCS in pTrcHis vector, same as pBAD-R, reverse primer Puro-F GCAACCTCCCCTTCTACGAGC...

...compare different clones of the same target to see if the results are the same/similar, emphasizing the antibody...your sample to. For example, we use our negative control to compare exposure times of our samples. A prominent...confirming it will produce the same results even to a different sample. Finally, as a lab we discuss the...underlying causes of this unreliability: cross-reactivity (binds to non-target proteins), variability (...Baker, 2015), causing a huge waste of precious samples, time, and funds – up to US$350 million in 2015...noise. We then compare the negative control to a sample with the primary included to confirm that the signal...antibody and not from the background noise. If the sample images similarly to the negative control, that ...

...particles = number of particles per mL of sample. The purity of the sample can be estimated by looking at the...Adenoviral vectors (AdV) are attractive vectors for research applications and gene therapy: they can ...Trapnell. "Evaluation of the concentration and bioactivity of adenovirus vectors for gene therapy." Journal...

...thiols, to cleave disulfide bonds. Load the samples, one sample per lane, onto the top of a resolving gel...all of the proteins in a sample migrate through the stacking gel at the same pace and enter the resolving...consistent size and density present in all samples. Uneven sample loading or incomplete protein transfer ...Figure 1: Overview of the Western blot process. The sample, a mixture of protein is separated in a gel, transferred...is the first step of a western. To prepare the samples for SDS-PAGE, measure the protein content and normalize...normalize to ensure equivalent loading. Denature the samples to their primary amino acid sequence by boiling...crosslinked polymer acrylamide. In addition to the samples, a protein standard of known molecular weight in...

...known titer on every qPCR run. Note that the rAAV sample has to be digested with DNase before undergoing...Grimm, D., Kern, A., Pawlita, M., Ferrari, F., Samulski, R., & Kleinschmidt, J. (1999). Titration of AAV...Harvey, B. K. (2017). Assaying the Stability and Inactivation of AAV Serotype 1 Vectors. Human Gene Therapy..., J., Hennigs, J. K., & Körbelin, J. (2021). Inactivation of Adeno-Associated Viral Vectors by Oxidant-Based...

...antibody signal in the retinal sample, but little to no signal in the liver sample, which you do (C), so your...epitopes on the same target reduces the likelihood that both antibodies would bind to the same off-target ...core goals of antibody validation:   Confirm reactivity with the target antigen - Recognition of an immunogen...to - particularly when you start using complex samples such as whole cell lysate or tissue sections. It...require variable expression of your target across samples - it is hard to distinguish between specific, invariable...there is a wealth of transcriptomic data from this sample type, so you use your bioinformatics skills (or...Removing (or reducing) the protein of interest in your sample, should lead to reduced or eliminated antibody ...

...pseudotype To pseudotype your viral vector, you use the same protocol for generating lentivirus, but you use ...Different pseudotyped viruses are differentially inactivated by nonspecific complement mechanisms in serum...anti-alphaGal antibodies. The VSV-G envelope protein is inactivated by serum regardless of the producer cell type...high concentrations as described above and is inactivated by the complement system in serum (DePolo et ...Vector Particles Produced in Human Cells Are Inactivated by Human Serum. Molecular Therapy 2:218–222 ....

... DIY Biology and, on occasion, would meet in the same space where BosLab exists today. Back then, it consisted...saw the potential in the space and decided to reactivate it. We started out by holding a few journal club... getting the community to collect various soil samples, extract DNA, sequence it, and analyze the data... characterize the microbiomes of the different samples. The goal is to not only learn something about ...