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... filters. Figure 1: Contamination! Tissue culture media often have indicators included in the media... difficult to eradicate. You can suppress the growth of mycoplasma but cannot eradicate it completely....important to prevent contamination rather than eradicating it later. Don’t be scared when mycoplasma infects...the first person to face this kind of problem. Educate others when you can and help them understand the...Cc, Drexler Hg (2002) Comparative Antibiotic Eradication of Mycoplasma Infections from Continuous Cell...

.... Channelrhodopsin-1 (ChR1) is excited by blue light and permits nonspecific cation influx into the cell...based on the LOV2 domain of Avena sativa phototropin 1 LOVETRAP reversibly sequester and release proteins...diversifying and extending optogenetics. Cell, 141 (1), 154–165. https://doi.org/10.1016/j.cell.2010.02.037...limitations and future developments. Exp Physiol, 96 (1), 19–25. https://doi.org/10.1113/expphysiol.2009.051961...Biol, 1408 , 141–165. https://doi.org/10.1007/978-1-4939-3512-3_10 PMID: 26965121 Yizhar, O., Fenno, L...Channelrhodopsins: cation channels from Chlamydomonas reinhardtii ChR2 Widely used light-gated cation channel from...light of the correct wavelength, the pore opens, cations flow into the cell (yellow dots), and the neuron...

... or coding strand of the transcribed gene (Figure 1). The coding strand is the DNA strand that encodes...that is transcribed by the RNA polymerase. Figure 1: Simplified promoter region during transcription. ...Mammalian Strong promoter from human elongation factor 1 alpha PGK Constitutive Mammalian Promoter from phospholycerate... Educational Resources Molecular Biology Reference Promoters...BioRender.com. DNA sequences called response elements are located within promoter regions, and they provide a stable...Promoters in bacteria contain two short DNA sequences located at the -10 (10 bp 5' or upstream) and -35 positions... (TSS) (Figure 2). The Pribnow box (TATAAT) is located at the -10 position and is essential for transcription...

...halorhodopsins to silence neuronal activity (Figure 1) (Wiegert et al., 2017). How to get an opsin into...and Jinfang Zhu. "Immunologic applications of conditional gene modification technology in the mouse." Current...contributed by Alessia Armezzani, scientific communication manager at genOway. A few decades ago, the brain...École Normale Supérieure de Lyon. Her love for educational science led her to pursue a teaching career, ..., and she now is a very happy scientific communication manager at genOway.     Acknowledgement:Through...Bacteriophage P1 site-specific recombination. Purification and properties of the Cre recombinase protein...): 7025-7030. PubMed PMID: 18614669. Belzung, Catherine, Marc Turiault, and Guy Griebel. "Optogenetics...

...Bachelor's degree Generally a short-term position (1-2 years) Often a gap-year position taken to transition...entire page dedicated to different career options from science policy to science communication and much ...), about their experiences. Research requires dedication and effort, so building a strong understanding...critically about whether it’s something you want to dedicate your time and energy to. How can I make the most... feel less confident in their abilities and qualifications. Studies on imposter syndrome have also found...universities often have scholarships and funds dedicated for undergraduates interested in doing research...the experience would improve my scientific and application writing skills, and ended up with the award. ...

...experience in cancer research After graduating with a 2:1 BSc in Molecular Biology (roughly a B average in the...at the Institute of Cancer Research in Sutton, located on the edge of London to gain relevant experience...recession. As each advertised job received many applications, employers were requesting that only scientists...commercial company. In my particular situation, communication between departments was sometimes difficult ... order to have meetings between teams. This communication is vital because otherwise I’d lose touch with...

... serve as origins of replication and also act as signals for packaging. Figure 1: Wild-type AAV genome...tissue tropism. Table 1 gives a summary of the tropism of AAV serotypes, indicating different serotype(s...AAV5, AAV8 Skeletal muscle AAV1, AAV8, AAV9 Table 1: Summary of tissue tropism displayed by different ...transgene, but also at a very low efficiency (less than 1% of wild-type). Another split AAV method which has...vectors pseudotyped with viral capsids from serotypes 1, 2, and 5 display differential efficiency and cell...potential formation of replication-competent viruses. While AAV themselves are replication incompetent, there...production of replication-competent viruses that would be able to infect cells and replicate autonomously...

...enzyme and guide RNA) for your experiment (Figure 1). You will decide how to express Cas9, the delivery...finally how to validate your genetic edit. Figure 1: Flow chart describing the general framework of a ...population. Some cells may remain wild type due to either (1) a lack of gRNA and/or Cas9 expression or (2) a lack...Biology , 1239 , 197–217. https://doi.org/10.1007/978-1-4939-1862-1_10 PMID: 25408407 Dixit, A., Parnas, O...Achieving your desired edit comes down to location, location, location. Positioning your chosen Cas enzyme ...designing the gRNA component for each category. Genetic Manipulation Application Cas Enzyme Additional Considerations...the table below, we highlight the most common categories of genetic manipulations used in mammalian cells...

... DNA fragments of interest, such as genes. Figure 1: Creation of recombinant DNA. Working with Plasmids...U169 recA1 endA1 hsdR17(rk-, mk+) phoA supE44 thi-1 gyrA96 relA1 tonA EPI300 LGC Biosearch Technologies...Addgene; Promega e14-(McrA-) recA1 endA1 gyrA96 thi-1 hsdR17(rK- mK+) supE44 relA1 Δ(lac- proAB) [F traΔ36...galK2 lacY1 proA2 rpsL20 (StrR ) xyl-5 λ- leu mtl-1 Top10 Invitrogen F- mcrA Δ(mrr-hsdRMS-mcrBC) Phi80lacZM15...Origin of Replication ( ori ) DNA sequence which directs the initiation of plasmid replication in bacteria... not located elsewhere in the plasmid backbone. In expression plasmids, the MCS is often located downstream...advantage of the process of DNA replication in vitro. During replication, the DNA helix is unwound by a...

...gamma-retroviruses ranges from 9–11 kb, encoded on RNA (Figure 1). This RNA is reverse transcribed into the provirus...producing viral vectors in a laboratory setting. Figure 1: Wild-type gamma-retrovirus genome. Created with BioRender.com...and tropism. Replication-competent The ability of a virus (or viral vector) to replicate and reproduce...hosts in order to continue their lifecycle and replicate. This feature can be harnessed for stable delivery... for safety to reduce the chance of creating replication-competent retroviruses. For a summary of all ... gamma-retroviral vectors integrate at random locations throughout the genome. Gamma-retroviral vectors...retroviral vectors are commonly used in gene therapy applications to more specifically direct delivery of gene...

...also negatively affect data reproducibility. Figure 1: Simplified flow chart for amplifying and using a ... Pooled Library Amplification Protocol for Addgene's generalized library amplification protocol. Read ...: Pooled Library Guide Introduction Library Amplification Types of Screens Resources Pooled libraries ..., semi-random sequences that can be used for applications like lineage tracing or parsing the effects ...of each cell carrying it as being a biological replicate for that plasmid. Library screens can be divided...

...NaH 2 PO 4 ∙H 2 O 1 L deionized water Adjust pH to 7.0 Autoclave or filter sterilize 1 M sodium phosphate...NaH 2 PO 4 ∙H 2 O 1 L deionized water Adjust pH to 7.0 Autoclave or filter sterilize 1 M of sodium phosphate...protease inhibitor cocktail. Add 1 part Protein A/G binding buffer to 1 part tissue culture supernatant...Choose Option 1 or Option 2 based on the concentration of the pooled sample above. Option 1: Buffer exchange...antibody to 1 mg/mL with PBS if needed. For long term storage, add sterile sodium azide to 1 mM. Option...purify recombinant antibodies. Workflow Timeline Day 1: Purify antibody Day 2 or later: Buffer exchange Equipment...°C bead bath Clamp stand and clamps Autoclave 0.1–1 mL single channel pipette 0.5–10 µL single channel...

...and placed in a buffer. Antibody Structure Figure 1: Structure of an antibody A standard antibody is made... together to form a “Y” shape, as shown in Figure 1. The two arms of the Y structure are responsible for...Handling Visualization Signal Amplification Application Overview Quantification Methods Capture Methods Visualization...Antibody Applications Antibody-based applications can be generally classed into four categories: Quantification... Read on to learn more about the applications common to each category, including an outline of the basic...these applications, visit the Antibody section of the Addgene Protocols page. Antibody Applications - Quantification...concentrates in the sample. Controls for quantification methods Quantification methods should be run with: a positive...

...Vented Falcon Tubes at 30-37 ℃, 225 rpm for 1 hour. After the 1 hour shaking period, pool and gently mix ...pulse and follow all specifications described in the equipment manual. Immediately add 1 mL SOC to cuvette...Last Update: August 17, 2023 Workflow Timeline Day 1: Transform, recover, set up overnight growth (Estimated... (Default: 4 tubes of Endura Duos, Lucigen, 60242-1) Alternatives include Stbl4 cells or other ultra-high...0.1 cm ) 20 mL SOC recovery media (Lucigen, 80026-1) 8X LB Agar + Antibiotic 245 mm bioassay plates (Molecular...into each of four 14 mL Vented Falcon Tubes and have 1 mL SOC per electroporation readily available for post-electroporation...autoclaved, sterile reagents for all steps. Procedure Day 1 Add 200 ng DNA to each 50 µL aliquot of thawed Endura...

...Preparation 1 M NaCl/PBS-MK buffer Dissolve 5.84 g of NaCl, 26.3 mg of MgCl 2 and 14.91 mg of KCl in 1× PBS ...iodixanol column gradient for AAV purification. Workflow Timeline Day 1: Purify Day 2: Buffer exchange and...cartoon indicating the position of the needle for harvesting of the purified AAV using option #1. Right...Dissolve 26.3 mg of MgCl 2 , and 14.91 mg of KCl in 1× PBS in a final volume of 100 mL. Sterilize by passing... step: mix 4.5 mL of 60% iodixanol and 13.5 mL of 1 M NaCl/PBS-MK buffer 25% iodixanol step: mix 5 mL ...Hamilton needle, taking care to avoid bubbles (Figure 1). 8 mL of 15% iodixanol step 5 mL of 25% iodixanol...disturb the gradient!** Collect Fractions Option #1 Prepare a row of roughly 20 open 1.5 mL microcentrifuge...

...more in our Restriction Cloning blog post . Figure 1: Restriction enzyme cloning of your gene of interest... Educational Resources Molecular Biology Reference Molecular...interest and then insert them into plasmids for replication and protein expression. There are many methods...restriction sites to any piece of DNA during PCR amplification, allowing for it to then be digested and ligated...cut your insert or backbone at only the desired location for your cloning project. Restriction enzyme cloning...transformed directly into E. coli , where the normal replication process will repair the nicks. It is important...

...of Contents A. pLKO.1-TRC Cloning Vector A.1 The RNAi Consortium A.2 Map of pLKO.1 A.3 Related plasmids...Order oligos compatible with pLKO.1 C. Cloning shRNA oligos into pLKO.1 C.1 Recommended materials C.2 Annealing... References H.1 Published articles H.2 Web resources I. Appendix I.1 Sequence of pLKO.1 TRC-Cloning Vector...information Back to Top A. pLKO.1-TRC Cloning Vector A.1 The RNAi Consortium The pLKO.1 cloning vector is the backbone...marker encoded in pLKO.1 allows for convenient stable selection. Figure 1 : Map of pLKO.1 containing an shRNA...puromycin should be from 1-10 μg/mL in 1 μg/mL increments. d. Label plates from 1-10 and add appropriate...Appendix I.1. Sequence of pLKO.1 TRC-Cloning Vector Click here to see the sequence of pLKO.1 TRC-cloning...

...DNA μL of 1 mg/mL PEI 1:1 18.9 18.9 1:2 18.9 37.8 1:3 18.9 56.7 1:4 18.9 75.6 1:5 18.9 94.5 1:6 18.9 113.4...μg total DNA to μg PEI ratios of 1:1, 1:2, 1:3 and 1:6. The 1:2 and 1:3 total DNA:PEI μg ratios provided...: Monday: Plate 1×10 6 cells in a T75 flask in 15 mL DMEM Complete. Wednesday: Plate 1×10 6 cells in a...Workflow Timeline Day 0: Seed 293T packaging cells Day 1 (pm): Transfect packaging cells Day 2 (am): 18 h post-transfection...inactivated in the lab by heating to 56°C for 30 min. 1 mg/mL polyethylenimine, linear MW 25,000 Da (PEI) ...to be empirically determined for each new batch of 1 mg/mL PEI and for each cell line. Considerations Before...enough PEI such that the ratio of μg DNA:μg PEI is 1:3 (1000 μL total per 10 cm dish). Using transfer plasmid...

...tissues: use 1 mL of Solution D per 100 mg of cells. For cultured cells: use 1 mL of Solution D per 1 X 10 7...tissues: use 1 mL of TRIzol® per 100 mg of cells. For cultured cells: use 1 mL of TRIzol® per 1 X 10 7 cells... to 1 mL of lysate: Add 0.1 mL of 2 M sodium acetate (pH 4.0), mix thoroughly by inversion. Add 1 mL water-saturated...Option A): Add 1 volume of Isopropanol to the extracted aqueous layer. Incubate at -20°C for 1 hour. Lithium.... Add 0.2 mL of Chloroform/Isoamyl alcohol (49:1) per 1 mL of TRIzol® used. Shake vigorously by hand for...or downstream applications. To improve yield of RNA, instead of incubating at -20°C for 1 hour, you can...

...Dilution 1:10 100 of Stock Virus 900 150 1,350 1:100 1:100 100 of 1:10 900 150 1,350 1:1,000 1:1,000 100...100 of 1:100 900 150 1,350 1:10,000 1:10,000 100 of 1:1,000 900 150 1,350 1:100,000 1:100,000 100 of 1... 1:10,000 900 150 1,350 1:1,000,000 Mix the dilutions well Note: the 1:10 dilution can usually be omitted...with 1 mL of 0.1% crystal violet for 10 min at RT. Gently remove the stain. Wash cells 3x with 1 mL of...dilutions. Sample Data Figure 1: A549 cells were transduced with the indicated serial dilutions of the lentiviral... media from the wells. Gently wash the cells with 1 mL of PBS and aspirate wash. Filter 0.1% crystal violet...volume in the well (mL) x dilution factor e.g., If the 1:100,000 well has 75 colonies, then there are 75 colonies...

...Volume of 1 mg/mL PEI (μL) 1:1 18.9 18.9 1:2 18.9 37.8 1:3 18.9 56.7 1:4 18.9 75.6 1:5 18.9 94.5 1:6 18.9...were transfected using 1:1, 1:2, 1:3 and 1:6 µg of pRosetta :µg of PEI. The 1:2 and 1:3 ratios provided high.... Thawed aliquots should be discarded after 1–2 months. 1 mg/mL polyethylenimine, linear MW 25,000 Da ...stable. Dilute 1:3 (µg DNA:µg PEI) in 500 µL total of OptiPro SFM (per 10 cm plate). 56.7 µL of 1 mg/mL PEI...subclone of HEK293T optimized for viral production) Day 1: Transfect Cells Day 2 (am): 18 h post transfection...to be empirically determined for each new batch of 1 mg/mL PEI prepared. There may be variation between...plasmid using a variety of ratios. Check the cells 1–2 days after transfection to determine what ratio ...

...dilution Dilution 1 (DNase step) 5 uL AAV stock 45 uL 10X 10X Dilution 2 5 uL Dil. 1 95 uL 20X 200X Dilution...range from 1 x 10 12 GC/mL to >2 x 10 13 GC/mL and we use an internal reference virus that is 1 x 10 13 ...dilutions, in duplicate, of your standard curve plasmid (2 x 10 9 stock made in step #1): Volume of 2 ...Data Figure 1: Example of a valid 8-point standard curve. Figure 2: Example of the amplification plots obtained...Equipment qPCR instrument Heating plate Pipettors 1–10 µL single channel pipette 20–200 µL single channel...channel pipette 200–1000 µL single channel pipette 1–10 µL multichannel pipette 2–50 µL multichannel pipette...The reference material should have a titer within 1-log of the expected titer of the samples being tested...

...Dilution 1 (20X): 5 µL in 95 µL 1X PCR buffer (1:20) Dilution 2 (20X): 5 µL in 95 µL 1X PCR buffer (1:400)...95 10 2 1 Denaturation 95 0.5 2 50 Annealing/Extension 60 1 2 50 Signal Stabilization 98 10 2 1 Hold 4 ...Use a single channel 1–10 µL pipette to add 5 µL of each viral sample to Dilution 1 in the 48-well dilution... 95 µL 1X PCR buffer (1:8,000) Dilution 4 (20X): 5 µL in 95 µL 1X PCR buffer (1:160,000) Dilution 5 (20X...95 µL 1X PCR buffer (1:3,200,000) Dilution 6 (2X): 50 µL in 50 µL 1X PCR buffer (1:6,400,000) Dilution ...50 µL 1X PCR buffer (1:12,800,000) Dilution 8 (2X): 50 µL in 50 µL 1X PCR buffer (1:25,600,000) Use multichannel...pipettes for the dilution series. For dilutions 1–5, use the 1–10 µL multichannel pipette set to 5 µL. For...

...7.5% Sodium Bicarbonate, 7.5 mL 1 M HEPES to 750 mL DMEM + 1 g/L glucose. 1 mg/mL polyethylenimine (PEI) ...Calculate the amount of each plasmid needed to have a 1:1:1 molar ratio with 2 mg total DNA per CS5 Plasmid ... determine the total μg/bp we need to achieve a 1:1:1 molar ratio of each plasmid: 2000 μg / 24,961 bp...deionized water + 100 mL of 1 M Tris HCl pH 8.5 + 60 mL of 5 M Sodium Chloride + 4 mL of 1 M Magnesium Chloride...magnesium, Corning 21-040-CV (cations can affect the attachment of adherent cells) 1 mg/mL Polyethylenimine ...completely. Combine all resuspended cell pellets and sonicate 5 x 1 sec pulses with at least 5 minutes on ice between...C before proceeding with the purification protocol. Sample Data Figure 1: HEK293T cells at various confluencies...

...from 1:1 to 1:6. Add the diluted PEI-MAX to the diluted DNA. Cap the tube and vortex with three 1 s pulses...solution at -20 °C. 1 mg/mL PEI-MAX Add 1 g of PEI-MAX powder to 900 mL deionized water in a 1 L bottle and ...to mix. Add 450 µL of 1 mg/mL PEI-MAX to the second tube of 6 mL BCD TFX (for a 1 mg/mL stock solution ...Last Update: February 18, 2022 Workflow Timeline Day 1: Seed cells Day 2: Transfect cells Day 3-6: Feed cells... a time. Add deionized water to a final volume of 1 L and recheck pH to ensure that it has not drifted...Aliquot and freeze upright at -20 °C. Procedure Section 1: Seeding cells The day prior to transfecting, seed...of two 50 mL tubes. Cap the tubes and incubate for 1 h in the 37 °C bead bath. Transfer the PEI-MAX and...

...both biosafety level 1 (BSL-1) and biosafety level 2 (BSL-2). The BSL-1 classification is for labs working...(PPE) for BSL-1 and BSL-2 labs. Protocols... Protocols Personal Protective Equipment (PPE) for BSL-1 and BSL-2 Labs...Labs Personal Protective Equipment (PPE) for BSL-1 and BSL-2 Labs Intro to the Lab Bench Check out more ...BSL-2 includes all of the precautions needed in BSL-1, however there are additional precautions that lie...always wear glasses/goggles in addition to the BSL-1 requirements. Conclusion Although simple, following...choose to reuse or repurpose this SOP in another location, please note that you do so at your own risk; ...

...polybrene (μL) 1:10 150 1348.5 1.5 1:25 60 1438.5 1.5 1:50 30 1468.5 1.5 1:75 20 1478.5 1.5 1:100 15 1483.5...the dilution factors (method 1) or the volume of virus (method 2): Method 1 Method 2 $$T = {N*F*D\over ...regulations. Workflow Timeline Day 0: Seed 293T cells Day 1: Transduce cells Day 2 (am): Remove media, replace...for 48–72 h. Gently aspirate media and replace with 1 mL of PBS. Calculate the fraction of fluorescent-positive... example: If 150,000 cells were transduced in the 1:100 well, resulting in 25% fluorescent cells, then...average of multiple dilutions. Sample Data Figure 1: 293T cells were transduced with a range of dilutions... these cells can still be used in downstream applications. Safety Warnings Lentivirus is generally considered...

...95 10 2 1 Denaturation 94 0.5 2 40 Annealing/Extension 60 1 2 40 Enzyme Deactivation 98 10 2 1 Hold 4 ... primers/probe (FAM) 1 µL 9 µL 900 nM, 250 nM 20X RPP30 primers/probe (HEX/VIC) 1 µL 9 µL 900 nM, 250 .... Last Update: July 7, 2023 Workflow Timeline Day 1: Seed and transduce cells Day 4: Treat cells with ...Cycler, Bio-Rad, T100 PCR Plate Sealer, Bio-Rad, PX1 1–10 µL single channel pipette 20–200 µL single channel...TrypLE, Thermo Fisher, 12605010) Ethanol, VWR, EX0276-1 Benzonase 250 U/µl, Millipore #71205-3 Polybrene 10... each viral dilution to a well of a 6-well plate (1 dilution per well). Leave one well untransduced (add...suspension well before seeding. Mix each well with a 1 mL pipette 5–10 times. The final volume in the well...

...June 14, 2023 Workflow Timeline Day 1: Run SDS-PAGE and stain gel Day 1 or later: Image analysis Video Watch...lane Example for AR0018 (lane 2 in Figure 1): Sample Peak 1 (contaminant) Peak 2 (contaminant) Peak 3 ...mobility of the bands (sample AR0016 in Figure 1) indicates that the samples may not have been processed...antibodies using Coomassie stain. Equipment Heat block 1–10 µL single channel pipette 2–20 µL single channel...deionized water. Gently invert to mix. Procedure Section 1: SDS-PAGE Add 5 µL of 4X sample buffer to each sample...attach to a power supply. Run the gel at 150 V for 1 h . Pro-Tip If the samples are running unevenly and...Add 20 mL of SimplyBlue SafeStain and incubate for 1 h with gentle agitation on a rocking platform. Pour...

... provides information for both biosafety level 1 (BSL-1) and biosafety level 2 (BSL-2). The purpose of...guidelines provide steps to ensure you are working in BSL-1 and BSL-2 labs safely. Protocols...Safety for Biosafety Levels One and Two (BSL-1 and BSL-2) Intro to the Lab Bench Check out more protocols...each level has different safety requirements. BSL-1 is designated for those working with microbes that...BSL-2 includes all of the precautions needed in BSL-1, along with additional precautions to prevent injuries...container Fire blanket Fire extinguisher Guidelines BSL-1 Guidelines Before You Work Right after entering the...hygiene officer. BSL-2 Guidelines Remember, the BSL-1 laboratory guidelines above are expected to be followed...