...ligates a promoter into an empty backbone can be repeated to ligate your gene of interest after the promoter...

... in the receptor and results in activation or repression of target gene expression. Nuclear receptors ...

...alkylguanine-DNA-alkyltransferase (hAGT), a DNA repair protein, and is suitable for both cell-permeant...

...entity is qualified to receive materials from our repository. All purchases are subject to Addgene’s Terms...

...) transforming protein SHOC2 SHOC2 leucine-rich repeat scaffold protein SOS SOS1 SOS2 SOS Ras/Rho guanine...

...expression vector encoding a split mCherry fluorescent reporter, enabling the detection of phosphorylation-dependent...

...for genome editing, as well as gene regulation (repression and activation). Genome support for bacteria ...

...TALENs for Endogenous Zebrafish Gene Targets Recent reports have shown that engineered TALENs can be used to...

...MiniPromoters MiniPromoter Source Gene Construct Reporter Negative Control N/A pEMS1312 cre N/A pEMS1301...

...FAP; mCerulean3 Marcel Bruchez 45944 pTDpelB-C_sfYFPTwinStrep Periplasmic space PelB signal sequence YFP...

... ensure representation is maintained. Maxipreps - Less is more: Do not overload the Maxipreps as yield...Conical tubes (Fisher, 14-432-22) Reagent Preparation Prepare, sterilize, and pour all LB Agar + Antibiotic...individual Maxipreps by Nanodrop, Picogreen, or Qubit. Each different DNA measurement type can report slightly...quantities of library for experimental applications. Repeated amplifications should be avoided as best as possible...and plasmid recombination can all impact the representation of individual plasmids in the pooled library...required for bacterial propagation (origin of replication and antibiotic selection). This recombination... high efficiency (1x10 10 cfu/µg) commercially prepared electrocompetent cells is strongly recommended...

...Self-inactivating long terminal repeat. f1 ori f1 bacterial origin of replication. Amp R Ampicillin resistance...pUC ori pUC bacterial origin of replication. 5’LTR 5’ long terminal repeat. RRE Rev response element. A.3...Avoid stretches of 4 or more nucleotide repeats, especially repeated Ts because polyT is a termination signal...AgeI. Mix: 6 μg pLKO.1 TRC-cloning vector (maxiprep or miniprep DNA) 5 μL 10x NEB buffer 1 1 μL AgeI add...use a miniprep kit to obtain DNA. 3. Conduct a restriction digest with EcoRI and NcoI: 1 μg miniprep DNA...transfection reagent. Replace with 5 mL fresh DMEM + 10% FBS + penicillin/streptomycin. Pipette the media...of this vector. A.2 Map of pLKO.1 pLKO.1 is a replication-incompetent lentiviral vector chosen by the TRC...

...polyethersulfone filter, Nalgene, 565-0010 (for viral preps, if prep was not previously filtered) Microcentrifuge...conical tubes, VWR 21008-216 Lentivirus preparation Reagent Preparation DMEM Complete: 10% v/v FBS and 4 mM...This protocol can be used to titer lentiviral preparations that confer antibiotic resistance. Protocols...This protocol can be used to titer lentivirus preparations that confer antibiotic resistance. Determining...Determining the titer of your lentiviral preparation allows you to control the multiplicity of infection (MOI...Timeline Day 0: Seed and transduce cells Day 2: Replace media with fresh media containing selection reagent... after 20–30 passages. Do not add penicillin/streptomycin to the media, as it can affect viability and...

...cells, increases reproducibility, as it eliminates the variation associated with repeated transient transfection...polyethersulfone filter, Nalgene, 565-0010 (for viral preps, if prep was not previously filtered) Microcentrifuge...long-term protein expression studies. Moreover, repeating experiments in a stable cell line, as opposed ...and transduce Cells Day 2–3 (am): Remove media, replace with fresh media containing selection reagent Day...15 mL conical tubes, VWR 21008-216 Lentivirus preparation Appropriate antibiotic for selection (e.g. puromycin...puromycin, blasticidin) Reagent Preparation DMEM Complete: 10% v/v FBS and 4 mM L-alanyl-L-glutamine (... after 20-30 passages. Do not add penicillin/streptomycin to the media, as it can affect viability and...

...the prep with full (genome containing) particles. On average, iodixanol-purified AAV preparations contain...ultracentrifugation to get pure adeno-associated virus (AAV) preps for use in your research. Protocols...and unlike other density gradient media, it is reportedly not necessary to remove iodixanol prior to use...separate out contaminants from an impure AAV preparation. The 15% iodixanol step has 1M NaCl to destabilize...iodixanol solution is available under the name OptiPrep (Link opens in a new window) from Sigma-Aldrich...Ultracentrifuge T70i rotor or equivalent Reagents Opti-Prep (60% Iodixanol) QuickSeal tubes QuickSeal tube spacers...Centrifugal filter units (MWCO 100 kDa) Reagent Preparation 1 M NaCl/PBS-MK buffer Dissolve 5.84 g of NaCl...

...determination. Prepare a 50:1 Reagent A to Reagent B dilution of the BCA assay. Prepare serial dilutions...the full details of our protocols supports reproducibility and accelerates science. Here, we list the ...solely for transparency intended to support reproducibility in science. General Guidelines Acrylamide percentage...Update: January 24, 2022 Workflow Timeline Day 1: Prepare lysates, run SDS-PAGE, transfer, block, incubate... Determine the total protein concentration and prepare the lysate for SDS-PAGE Determine the protein concentration...need to load a greater amount of total protein. Prepare sample for loading as follows: Add 10% v/v β-mercaptoethanol...samples for 10 min at 100 °C . Section 3: SDS-PAGE Prepare the precast gel as follows: Remove the gel from...

...polyethersulfone filter, Nalgene, 565-0010 (for viral preps, if prep was not previously filtered) Microcentrifuge...conical tubes, VWR 21008-216 Lentivirus preparation Reagent Preparation DMEM Complete: 10% v/v FBS and 4 mM...Protocol that can be used to titer lentiviral preparations carrying a fluorescence marker. Protocols...This protocol can be used to titer lentiviral preparations that carry a fluorescence marker. Determining...Day 1: Transduce cells Day 2 (am): Remove media, replace with fresh media Day 4–5: Count fluorescent cells... after 20–30 passages. Do not add penicillin/streptomycin to the media, as it can affect viability and... 75,000 cells into each well of a 6-well dish. Prepare a batch of cells as follows: Dilute 525,000 cells...

...FAM-CACTCCCTCTCTGCGCGCTCG-BBQ- Reagent Preparation 1X PCR Buffer containing 0.05% Pluronic F-68: Prepare immediately before...cool. Prepare the ITR master mix in a microcentrifuge tube as shown below. For 8 samples prepare enough...GC/mL, where GC is genome copies and generally represents the physical number of viral particles. Users...4475 µL Molecular Biology Grade Water Procedure Preparation Before handling any viruses, get materials ready...sufficient pipette tips and reagent reservoirs. Pro-Tip Prepare the viral dilutions in a separate biological safety...(dilution BSC) than that used for master mix preparation and droplet generation (droplet generation BSC...well plate sealer by gently touching the screen. Prepare the Serial Dilution Place a 48-well dilution plate...

...the full details of our protocols supports reproducibility and accelerates science. Here, we list the ...solely for transparency intended to support reproducibility in science. General Considerations For antibody-expressing...antibody-expressing plasmids containing the SV40 origin of replication use a HEK293 line stably expressing the SV40 ... containing the Epstein-Barr virus origin of replication use a HEK293 line stable expressing Epstein Barr...BCD TFX) to 37 °C. See below for other Reagent Preparation instructions. Wipe down all pipettes and equipment...equipment with 10% bleach prior to use. Reagent Preparation 100 mM Valproic Acid Dissolve 2.88 g valproic...Sterilize by passage through a sterile 0.22 µm filter. Prepare 10 mL aliquots and store the solution at -20 °C...

...prestained protein ladder on ice. Reagent Preparation Prepare IgG Standard Dilute the 2.5 mg/mL IgG isotype...the full details of our protocols supports reproducibility and accelerates science. Here, we list the ...solely for transparency intended to support reproducibility in science. Last Update: June 14, 2023 Workflow.... Add 8 µL of PBS to a total volume of 13 µL . Prepare the recombinant antibody Dilute or concentrate ...microfuge tube. Add 8 µL of PBS to total 13 µL . Prepare 1X MOPS buffer Dilute 25 mL of 20X MOPS buffer ...appropriate imaging system. Figure 1 Recombinant antibody preps should have 2 clear bands at ~50 kDa and ~25 kDa...Analyze . Select Gels . Select Select Next Lane . Repeat for all lanes. Plot the area under the curves for...