...Automated cell counter 37 °C, 5% CO 2 incubator with shaking platform set to 120 rpm 37 °C bead bath Vortex Stir...: Harvest antibody Equipment Biosafety Cabinet 4 °C Refrigerator Micropipettes and tips Pipettor and pipettes...BalanCD HEK293 transfection media (BCD TFX) to 37 °C. See below for other Reagent Preparation instructions...Prepare 10 mL aliquots and store the solution at -20 °C. 1 mg/mL PEI-MAX Add 1 g of PEI-MAX powder to 900 ....22 µm filter. Prepare aliquots and store at -20 °C until use. BCD TFX 1000 mL BalanCD HEK293 Media 10...Glutagro Do not add selective reagents Store at 4 °C until use. We suggest preparing fresh solutions after...BalanCD HEK293 Feed 20 mL 200 mM Glutagro Store at 4 °C until use. We suggest preparing fresh solutions after...

...min at 4 °C and discard the flow through. Add your sample. Spin at 3500 rpm for 5–8 min at 4 °C, discard...possible. Store at 4 °C for short term (2 weeks), or aliquot and store at -80 °C for long term. Sample... passing through a 0.22 μm filter and store at 4 °C. 1X PBS-MK buffer Dissolve 26.3 mg of MgCl 2 , and... passing through a 0.22 μm filter and store at 4 °C. 0.1% Pluronic-F68 in PBS (A) Add 500 µL of 10% Pluronic-F68... mL PBS 0.001% Pluronic-F68 in PBS + 200 mM NaCl (C) (formulation buffer) Add 5 mL of Buffer B and 2 mL... at 350,000 x g for 90 min in a T70i rotor at 10 °C. Pro-Tip If you need more time, you can alternatively...alternatively centrifuge for 2 h at 200,000 x g at 18 °C. Carefully take the QuickSeal tubes out of the rotor ...

...Shaking incubator at 37 °C Stationary incubator at 37 °C Water bath at 42 °C Ice bucket filled with ice... storage at 4°C and let warm up to room temperature and then (optional) incubate in 37°C incubator. Mix...transform Procedure Take competent cells out of -80°C and thaw on ice (approximately 20-30 mins). Remove...placing the bottom 1/2 to 2/3 of the tube into a 42°C water bath for 30-60 secs (45 secs is usually ideal...without antibiotic) to the bacteria and grow in 37°C shaking incubator for 45 min. Pro-Tip This outgrowth...and won't grow in colonies. Incubate plates at 37°C overnight. Tips and FAQ How can I save time when carrying.... Transformation of bacteria with plasmids is important not only for studies in bacteria but also because...

...instrument using SYBR detection: 98 °C 3 min / 98 °C 15 sec / 58 °C 30 sec / read plate/ repeat 39x from...and store at -20 °C. Once a standard is thawed do not freeze it again but store at 4 °C and use within 1...dilution. References Aurnhammer C, Haase M, Muether N, Hausl M, Rauschhuber C, Huber I, Nitschko H, Busch ...mix sample (do not vortex) Incubate 30 min at 37 °C Transfer to ice ** Critical: do NOT treat your plasmid...10 7 1.00 x 10 6 1.00 x 10 5 1.00 x 10 4 empty NTC C AAV reference Sample 3 D E Sample 1 Sample 4 F E Sample...presence of a second peak at a temperature of ~70–75 °C usually indicates the presence of primer dimers which...vector cores. Our titers were similar to those reported by these institutions. This protocol is for a ...

... Store the tubes at -80 °C. After thawing, the solution can be stored at 4 °C for up to 2 months. After.... Add stir bar and stir slowly at 4 °C for 1 h, then keep at 4 °C for 3 h without stirring to allow full...it can be inactivated in the lab by heating to 56 °C for 30 minutes. 0.45 μm polyethersulfone (PES) filter...11 mL of 200 mM L-alanyl-L-glutamine. Store at 4 °C. D1 + 0.1 M sorbitol (optional) : DMEM low glucose...may separate into phases. Aliquot and store at 4 °C. Cell Lysis Buffer : 50 mM Tris HCl, 150 mM NaCl,...sterilize through a 0.22 μm membrane. Store at 4 °C. Considerations Before You Start The health of the...and cells. Centrifuge at 3900 rpm for 20 min at 4 °C to pellet the cells. Keep the cell pellet on ice and...

...Store the tubes at -20 °C or -80 °C. After thawing, the solution can be stored at 4 °C for up to 2 months....heat-inactivated FBS and 5 mL of glutaGRO. Store at 4 °C. Pro-Tip Different brands and lots of FBS can promote...it can be inactivated in the lab by heating to 56°C for 30 min. 1 mg/mL polyethylenimine, linear MW 25,000... tissue culture plates. Incubate the cells at 37 °C, 5% CO 2 for ~20 h. Prepare a mixture of the 3 transfection...media to a polypropylene storage tube and store at 4 °C between harvest. Centrifuge the viral supernatant ...filter. The viral supernatant can be stored at 4 °C for several hours but should be aliquoted and snap...snap frozen in liquid nitrogen and stored at -80 °C as soon as possible to avoid loss of titer. Sample Data...

...bases 40-60% G/C content Start and end with 1-2 G/C pairs Melting temperature (Tm) of 50-60°C Primer pairs...pairs should have a Tm within 5°C of each other Primer pairs should not have complementary regions Note:...toward one another. The size of the primer is very important as well. Short primers are mainly used for amplifying...

... PMID 22341318 Gradinaru V, Zhang F, Ramakrishnan C, Mattis J, Prakash R, Diester I, Goshen I, Thompson...20621963 Mattis J, Tye KM, Ferenczi EA, Ramakrishnan C, O'Shea DJ, Prakash R, Gunaydin LA, Hyun M, Fenno ...neural populations at distinct times. Temporal considerations. Temporal precision is key in optogenetic experiments... exposed to light of the correct wavelength, the pore opens, cations flow into the cell (yellow dots),...optical switches Sensors are genetically-encoded reporters of molecular signals; e.g., calcium indicators...specific subsets of neurons, allowing precise spatiotemporal control of these neurons by turning on and ...addition of trafficking signal from Kir2.1 and ER export signal provide improved membrane targeting 589 ...

...hours) at 37 °C. Pro-Tip Some plasmids or bacteria need to be grown at 30 °C instead of 37 °C. This is often...unstable plasmids, which sometimes recombine at 37 °C. Be sure to check this before incubating your plate...initials. Labeling within a laboratory setting is important for organization, and it is recommended that you...

... incubating certain steps at 4 °C instead of room temperature or 37 °C. The protocol notes when there ...well plate seal and incubate at 37 °C for 30 min , or overnight at 4 °C . Section 2: Block the plate Prepare...rpm for 2 h at room temperature or overnight at 4 °C . Section 3: Primary antibody incubation Carefully...rpm for 2 h at room temperature or overnight at 4 °C . Section 4: Secondary antibody incubation Carefully...rpm for 2 h at room temperature or overnight at 4 °C . Section 5: TMB reaction Carefully remove the plate...that sharing the full details of our protocols supports reproducibility and accelerates science. Here,...information is solely for transparency intended to support reproducibility in science. General Considerations...

...Spatula Platform shaker Cold room Gel imager -80 °C freezer Reagents 1X PBS Lysis buffer e.g., RIPA lysis... Centrifuge lysate for 15 min at 14,000 x g at 4 °C . Transfer supernatant to a clean microcentrifuge ...tube. Use the lysate immediately or store at -80 °C until ready to use. Section 2: Determine the total...well microtiter plate. Incubate for 30 min at 37 °C . Determine the absorbance at 590 nm . Calculate the...sample to 1X . Boil the samples for 10 min at 100 °C . Section 3: SDS-PAGE Prepare the precast gel as follows... the membrane overnight in primary antibody at 4 °C on a rocking platform. Pro-Tip Primary antibody incubation...that sharing the full details of our protocols supports reproducibility and accelerates science. Here,...

...spectrophotometer 37 °C, 5% CO 2 incubator with shaking platform set to 120 rpm 37 °C bead bath Clamp stand...Equipment Class II, Type A2 Biological Safety Cabinet 4 °C Refrigerator Pipette controller Benchtop centrifuge...phosphate dibasic (NaH 2 PO 4 ) Store up to 1 month at 4 °C Adjust pH to 7.0 Autoclave or filter sterilize Protein...µm PES filter. Aliquot and freeze upright at -20 °C Procedure Section 1: Affinity chromatography Equilibrate...A/G binding buffer and store in 20% ethanol at 4 °C . Pro-Tip Columns may be re-used up to 5x when purifying...446 Benzamide, Millipore Sigma 12072 Antipain, Millipore Sigma 10791 Leupeptin, Millipore Sigma L2884 Aprotinin...monobasic monohydrate, Millipore Sigma 71507-250G Sodium phosphate dibasic, Millipore Sigma S7907-500G 1 ...

...at 37°C for 12-18 hr in a shaking incubator. Note: Some plasmids or strains require growth at 30°C. If ...more slowly. Also, bacteria incubated at 30°C rather than 37°C often require longer incubation times. Double...plasmid. However, a liquid culture is capable of supporting a higher density of bacteria and is used to grow...

...parameters: Cycling Step Temperature (°C) Time (min) Ramp Rate (°C/sec) # Cycles Enzyme Activation 95 10...titer: $$T = {V*C*D\over v}$$ Where: T = Infectious titer, TU/mL V = Viruses per genome C = # Cells per ...Bio-Rad, 1863051 8-strip PCR tubes, Axygen, PCR-02-FCP-C ddPCR 96-well PCR plates, Bio-Rad, 12001925 Pierceable...Benzonase in DMEM complete to each well. Incubate at 37 °C for 30 min. Gently aspirate media from wells, using...can be used for ddPCR immediately or stored at -20 °C until ready to use. Preparing for ddPCR Thaw samples...EX0276-1 Benzonase 250 U/µl, Millipore #71205-3 Polybrene 10 mg/mL, Millipore, TR-1003-G Molecular Biology...place the PCR plate with the foil onto the metal support block. Place the block in the plate sealer and ...

...attention. Let agarose solution cool down to about 50 °C (about when you can comfortably keep your hand on ... with a pipette tip. Place newly poured gel at 4 °C for 10-15 mins OR let sit at room temperature for ... set more quickly if you place the gel tray at 4 °C earlier so that it is already cold when the gel is...period of time; b) using a wider/thinner gel comb; or c) loading less DNA into the well. Another method for... be in the bottom portion of the gel, but all of the EtBr will be in the top portion and your bands will...overboil the solution, as some of the buffer will evaporate and thus alter the final percentage of agarose...

...individual tubes of 5, 10 or 20μL for storage at -20°C. Whenever you need to set up ligations in the future...optimization. Incubate at room temperature for 2hr, or at 16°C overnight (following the manufacturer’s instructions...efficiency of ligation can be improved by incubation at 37°C. Proceed with bacterial transformation . Tips and ...desired insert. If you are in this situation, it is important to treat the digested vector backbone with a phosphatase...setting up the ligation reaction itself, it is important to determine the amount of cut insert and vector...being sure to increase the amount of buffer proportionally. 1μL of ligase should be sufficient for larger...

...heat-inactivated FBS and 5mL of 100X glutaGRO. Store at 4 °C. Pro-Tip Different brands and FBS lots can promote...it can be inactivated in the lab by heating to 56 °C for 30 min. Considerations Before You Start The health...dose of antibiotic, which may not be stable at 37 °C. To achieve a stable cell pool, the antibiotic selection...well plate and left undisturbed for 13 days. (a, b, c) Colonies formed by expansion of single cells for ...CI) Heat-inactivated FBS Polybrene (10 mg/mL), Millipore TR-1003-G 1X PBS pH 7.4 without calcium or magnesium...of cell death upon antibiotic selection. It is important to monitor these cells regularly and replace the... the surviving cells in the culture, so it is important to do regular media changes and maintain optimal...

... Cepko, 2020). Incubate the mix for 1 hour at 50 °C or follow manufacturer's instructions. You can purchase...Gibson DG, Smith HO, Hutchison CA, Venter JC, Merryman C. (2010). Chemical synthesis of the mouse mitochondrial....1515 (Link opens in a new window) Rabe BA, Cepko C. (2020). A Simple Enhancement for Gibson Isothermal...other. Phusion High-Fidelity DNA Polymerase - incorporates nucleotides to “fill in” the gaps in the annealed... product by restriction digest . Sequence the important regions of your final plasmid, particularly the...

...parameters. Cycling Step Temperature (°C) Time (min) Ramp Rate (°C/sec) # Cycles Denaturation 95 10 2 1 ...sample: $$T = {R*C*D*1000\over V}$$ Where: T = Titer, GC/mL R = Reaction volume (20 µL) C = Copies/µL D ...Bio-Rad, 1863051 8-strip PCR tubes, Axygen, PCR-02-FCP-C ddPCR 96-well PCR plates, Bio-Rad, 12001925 48-well...place the PCR plate with the foil onto the metal support block. Place the block in the plate sealer and ... reading. When the droplet reading is complete, export the data from all wells as a CSV file and use the...

...different regions of the central nervous system. Burger C, Gorbatyuk OS, Velardo MJ, Peden CS, Williams P, Zolotukhin...lentiviruses integrate into the genome. This is important because local chromatin structure at the site ...

...) may be more efficacious than gRNAs containing a C nucleotide at the same position in spite of being ...promoter is typically used for gRNA May contain reporter gene (e.g. GFP) to identify and enrich positive...vector or separate transfer vectors May contain reporter gene (e.g. GFP) or selection marker to identify... to target cells through microinjection or electroporation Transient expression of CRISPR components Expression...may alter protein function when they occur in important protein domains. For gene editing experiments ... addition to off-target activity , it is also important to consider factors that maximize cleavage of ...25408407 Hashimoto, M., & Takemoto, T. (2015). Electroporation enables the efficient mRNA delivery into the...

...heat-inactivated FBS and 5 mL of 100X glutaGRO. Store at 4 °C. Pro-Tips Different brands and lots of FBS can promote...it can be inactivated in the lab by heating to 56 °C for 30 min. Considerations Before You Start The health...CI) Heat-inactivated FBS Polybrene (10mg/mL), Millipore TR-1003-G 1X PBS pH 7.4 without calcium or magnesium...

...media at 37 °C (for at least 15 minutes). Prewarm 3X LB Agar + Antibiotic plates at 37 °C. Prewarm 8X ...Chill a box of 200 µL micropipette tips in a -20 °C freezer. Aliquot 3 mL SOC into each of four 14 mL ... mix. Electroporate cells (one at a time for a total of eight electroporations): Electroporator Conditions...and have 1 mL SOC per electroporation readily available for post-electroporation recovery of cells. Ensure...if the recombinant band makes up a significant proportion of the DNA pool. Last Update: August 17, 2023...limited. Equipment Table top centrifuge BioRad Electroporator (MicroPulser TM , Bio-Rad 1652100) Reagents.../or increasing the skewness of the pool. 8 electroporation cuvettes (BioRad, Micropulser, 0.1 cm ) 20 ...

...HIV-1 Genome Nuclear Import Is Mediated by a Central DNA Flap. Zennou V, Petit C, Guetard D, Nerhbass ...and systems. When planning your experiment, the important component to consider and optimize is the transfer...Cre-based regulation and fluorescent fusions or reporters. Browse lentivirus plasmids available from Addgene...order to create functional lentiviral particles. IMPORTANT : A 3rd generation transfer plasmid can be used...cells, they kill off any cells that have not incorporated the lentiviral genome and those cells that survive...expanded to create stable cell lines, which have incorporated the lentiviral genome and harbor the genetic...interest and contains the sequences that will incorporate into the host cell genome, but cannot produce...

...inactivated FBS and 5 mL of 100X glutaGRO. Store at 4 °C. Pro-Tip Different brands and lots of FBS can promote...it can be inactivated in the lab by heating to 56 °C for 30 min. Considerations Before You Start The health...CI) Heat-inactivated FBS Polybrene (10 mg/mL), Millipore TR-1003-G 1X PBS pH 7.4 without calcium or magnesium...

...standard Before Starting Warm the hot plate to 100 °C. Thaw IgG standard and prestained protein ladder on...microcentrifuge. Heat the samples for 10 min at 100 °C in a heat block. Spin the sample briefly in the microcentrifuge...IgG standards in Google Sheets. Open exported CSV file. Select import. Choose file. Format as needed. Select...that sharing the full details of our protocols supports reproducibility and accelerates science. Here,...information is solely for transparency intended to support reproducibility in science. Last Update: June 14...intensity of the protein bands to the overall lane. Import the gel image into ImageJ. Select File . Select... each peak by selecting the center of the peak. Export results as a csv file. In the results table, select...

...longer period of time; b) using a wider gel comb; or c) loading less DNA in the well. How do you get better...around the band as possible. To do so, it is often important to take the excised band, lay it down on the UV...sides with the razor blade. This is especially important during the DNA purification step, as many kits... fragment. The weight of the gel is directly proportional to its liquid volume and this is used to determine...manufacturer's instructions. Note: It is usually important to determine the concentration of the DNA that...

... refrigerators to achieve a temperature between 4°C and room temperature. This general protocol suits ...on the surfaces of the water bath as the water evaporates from the tub. Disinfectant may be added to prevent...cover on the top of the water bath to prevent evaporation and maintain the desired temperature. This also...

...software to ensure a melting temperature between 50-60°C for your PCR primers. Step 2: Linearize Vector In ...provided by your polymerase manufacturer. It is very important to remove all free nucleotides from your PCR product...