...for transfecting mammalian cells using linear polyethylenimine. Transfections allow for transient expression...Different brands and lots of FBS can promote or inhibit transfection. Test a variety of brands and lots...RT. Carefully transfer the transfection mix to your HEK293T cells. Add the transfection mix dropwise, ...viral production) Day 1 (pm): Transfect Cells Day 2 (am): 18 h post transfection - Remove media, replace with...protocol to transfect mammalian cells with your plasmid of interest. Protocols... Protocols General Transfection General...General Transfection You may also like... Viral Vector Guides Virus Blog Posts Mol Bio Protocols Viral Service...

...Introduction Transformation is the process by which foreign DNA is introduced into a cell. Transformation of bacteria... more easily transformed and that will help to maintain the plasmid without rearrangement of the plasmid...prepared for optimal transformation efficiencies upon thawing. For the highest transformation efficiency, we...* Commercial competent cells range significantly in their transformation efficiency. The lowest efficiency...time when carrying out transformations? If you are not concerned with transformation efficiency (such as ... 20-30 mins) I get very few if any transformants when transforming large plasmids (>10 kb) or BACs, what...Learn how to transform E. coli with your plasmid of interest. Protocols...

... to proceed with transfection. Transfect the flask containing 108 mL cells: Transfer 6 mL of BCD TFX into...General Transfection Protocol Recombinant Antibody Purification Protocol Introduction Transfections allow...using Polyethylenimine Max as a transfection reagent. After transfection and expression, the recombinant...Section 2: Transfection Check the cell density and viability: Using a 5 mL pipette, transfer 0.5 mL of ...feeding schedule: Thursday: Transfect cells. Friday (24 h post-transfection): Add 4% BCD Feed. Monday (...post-transfection): Add valproic acid to 3.75 mM, add 4% BCD Feed. Tuesday (120 h post-transfection): Add...Wednesday (144 h post-transfection): Add 4% BCD Feed. Thursday (168 h post-transfection): Harvest antibody...

...Different brands and FBS lots can promote or inhibit transfection. Test a variety of brands and FBS lot...-term protein expression observed using transient transfection approaches, generating cell lines using...experiments in a stable cell line, as opposed to transiently-transfected cells, increases reproducibility, as it... the variation associated with repeated transient transfection. Some lentiviral vectors deliver mammalian...stable cell culture after transduction. Performing antibiotic selection on transduced cells enables elimination...Pro-Tip* Transducing too many cells relative to the number of virus particles reduces the transduction efficiency... may have 1 copy of the transgene, while others have >1 copy of the transgene in their genome. This can...

...Gel Transfer Device. Wet the pre-run gel in deionized water and place it on the transfer membrane of the...protocol uses a dry transfer device but can be adapted for wet and semi-dry transfer approaches. Last Update...well. Assemble the transfer sandwich as follows: Unseal the iBlot 2 PVDF Mini transfer stack. Set the Top... parameters are correct. Transfer for 5–6 min for proteins <30 kDa. Transfer for 8–10 min for proteins...before running your experiment. Remove the membrane from the transfer device and place it in a container with...Block the membrane in blocking buffer for 1 h at RT on a shaking platform. Wash the membrane 3x for 5 ...Incubate the membrane with secondary antibody for 60 min at RT on a shaking platform. Wash the membrane 3x for...

...Different brands and lots of FBS can promote or inhibit transfection. Test a variety of brands and lots...lentiviral vector transfected into 293T cells using a polyethylenimine (PEI) transfection protocol. This ...packaging cells Day 1 (pm): Transfect packaging cells Day 2 (am): 18 h post-transfection. Remove media, replace...optimized for each transfer plasmid. *Pro-Tip* Endotoxins can inhibit transfection; therefore, plasmid...modified for alternative packaging cell lines or transfection reagents. Once produced, lentivirus can be used...hydroxide 0.22 μm polyethersulfone (PES) filter Transfection grade DNAs Reagent Preparation DMEM Complete...drifted. Filter the solution through a 0.22 μm membrane. Aliquot 500–1000 μL into sterile tubes. Store...

...drives RNA Polymerase III transcription for generation of shRNA transcripts. cPPT Central polypurine tract...HIV-derivative viruses. For transient knockdown of protein expression, you may transfect plasmid DNA directly...Day 2: b. Perform the transfection in the late afternoon because the transfection mix should only be incubated...it, and does not grant any license, express or implied, under any patent. All transfers of materials from...protocol is provided for your convenience. See “warranty information” in appendix. Table of Contents A....Digesting pLKO.1 TRC-Cloning Vector C.4 Ligating and transforming into bacteria D. Screening for Inserts D.1 Recommended...Sequence of pLKO.1 TRC-Cloning Vector I.2 Recipes I.3 Warranty information Back to Top A. pLKO.1-TRC Cloning ...

... volume of homogenized cell solution is transferred. Transfer cells from the homogenized cell solution...990 µL complete medium. Instead of transferring 0.125 µL, now transfer 100 times that volume, which is 12.5... solution, transfer 12.5 µL of the 1:100 dilution to 10 mL of conditioned medium. Transfer 100 µL of the...generated from A549 cells transduced with lentiCas9-Blast 1 . A549 cells were transduced (MOI = 37) and selected...line from a polyclonal pool of stable cells. Transducing cells with lentivirus results in a heterogenous...heterogeneous cell pool could lead to reduced transgene expression over time, as the lower expressing ...populations that are more likely to retain stable transgene expression. Other methods of generating a monoclonal...

...Different brands and lots of FBS can promote or inhibit transfection. Test a variety of brands and lots...cells in CS2 Day 2: Seed cells in CS5 Day 3 (am): Transfect cells Day 7 (am): Harvest cells Equipment Class...Polyethylenimine (PEI) 25 kDa MW *Pro-Tip* Other transfection reagents may be used in this protocol, but their...their conditions must be optimized. Plasmids for transfection: pHelper; pRC (Rep-Cap), plasmid expressing ...drifted. Filter the solution through a 0.22 μm membrane. Aliquot 500–1000 μL into sterile tubes. Store... pH to ~8.5 Filter sterilize through a 0.22 μm membrane. Store at 4 °C. Considerations Before You Start... Pass and plate the cells the day before the transfection. Thaw a new vial of cells after 30 passages....

...Different brands and lots of FBS can promote or inhibit transfection. Test a variety of brands and lots...the same time as the viral transduction. For some cell lines, transduction is optimized if the cells are...When the untransduced (negative control) cells have died and colonies are visible in the transduced wells...June 16, 2016 Workflow Timeline Day 0: Seed and Transduce Cells Day 2: Replace media with fresh media containing... are seeded the day before viral transduction. Note that in that case, polybrene should only be added ...added at the time of viral transduction, and not during the cell seeding step. Procedure Before beginning ...empirically determined. Treat the target cells with a range of doses of antibiotic. Determine the minimum concentration...

... is based on an LV titer range of 1E+05–1E+09 TU/mL, where TU is transducing units and generally represents...RPP30) to determine the level of transduction. However, cells transduced with lentivirus may receive one...July 7, 2023 Workflow Timeline Day 1: Seed and transduce cells Day 4: Treat cells with Benzonase and harvest...Complete 3 µL of 250 U/µL benzonase Procedure Transducing Cells Thaw the virus on ice. Prepare the following... pipette 10–20 times. Use a new pipette tip to transfer 200 µL of the first dilution into the next dilution... plate (1 dilution per well). Leave one well untransduced (add 150 µL of DMEM complete instead of a viral... into the wells containing the virus and the untransduced control. *Pro-Tip* For even seeding, prepare...

...Different brands and lots of FBS can promote or inhibit transfection. Test a variety of brands and lots... were transduced with a range of dilutions of pHAGE-TO-dCas9-3XmCherry . 72 h post transduction, cells...cells transduced F = Fraction of cells with fluorescence D = Dilution Factor V T = Transduction Volume...Workflow Timeline Day 0: Seed 293T cells Day 1: Transduce cells Day 2 (am): Remove media, replace with fresh...1468.5 1.5 1:75 20 1478.5 1.5 1:100 15 1483.5 1.5 Untransduced 0 1498.5 1.5 Mix the dilutions well Gently aspirate... one dilution with one well remaining as the untransduced control). Count the cells in the remaining well...underestimation of the true titer. Calculate the transduction units per mL (TU/mL) using either the dilution...

...step heats the double stranded DNA template strand to the point where the strands start denaturing and ...this temperature range to anneal to each of the single stranded DNA template strands. The DNA polymerase...of the sense and anti-sense strands of the template DNA. They are strands of nucleic acid that are starting...molar ratio. It also requires two unique single stranded DNA oligonucleotide (oligo) primers, which anneal...seconds at 94°C: Continued denaturation of double stranded DNA. Anneal primers for 30 seconds at 55°C: The...polymerase to synthesize and elongate the new target DNA strand accurately and rapidly. Repeat steps 2-4 25-30 ...fill-in any protruding ends of the newly synthesized strands. Materials List Reagents for each 50µL PCR : Thin-walled...

... Day 1: Transform, recover, set up overnight growth (Estimated time 2-3 hours) Transformation should be...of 5 mL (3 mL SOC + 2 mL transformed Endura from two separate transformations). *Pro-Tip* Do not pipette...assumes familiarity with standard bacterial transformation and basic knowledge of the library being amplified...amplified. Please see our protocol on Bacterial Transformation . The generalized pooled library amplification...premixed LB (VWR 101414-072) for convenience but any brand or house- made LB that supports normal growth is...using a new vented falcon tube for every two transformations. Each of the four 14 mL Vented Falcon Tubes...repeatedly or mix when removing SOC containing transformed DNA-Endura from cuvette. Shake four 14 mL Vented...

...incubation, transform DH5α E. coli cells by heatshock with the plasmid of interest. See our transformation page... for a detailed heatshock transformation protocol Plate 50 µL of transformed E. coli /rescue media suspension...also like... Making LB Agar Plates Bacterial Transformation Recovering Plasmid DNA from Bacterial Culture...plating antibiotic over-agar for the selection of transformed E. coli . Over-agar spreading of antibiotic makes...investigator to conveniently plate and select transformed cells containing plasmids differing in their ... sterile water (or other antibiotic) E. coli transformed with a plasmid containing the carbenicillin (...carbenicillin, plated over-agar for selection of transformed E. coli . Sample Data Selection of E.coli on ...

...recipient plasmid backbone. Transformation: Proceed with the transformation according to the manufacturer...from your transformation will give you the first indication as to whether your transformation worked. Your...Digest of Plasmid DNA DNA Ligation Bacterial Transformation Summary PCR based cloning is incredibly versatile...chance for mis-priming, so you can use a pretty wide range of annealing temperatures, but you may need to increase...competent cells. For most standard cloning, you can transform 1-2μl of your ligation reaction into competent...conduct a positive control to ensure that your transformation worked. You could also try varying the amount...cloning. DNA replication by PCR has error rates that range from roughly 1 per 500bp to roughly 1 per 10 million...

...-ligating recipient plasmid backbone. Transformation: Transform your ligation reaction into your bacterial...from your transformation will give you the first indication as to whether your transformation worked. our...Digest of Plasmid DNA DNA Ligation Bacterial Transformation Summary The following technique can be used...competent cells. For most standard cloning, you can transform 1-2μl of your ligation reaction into competent...conduct a positive control to ensure that your transformation worked. You should also verify that you are...

...now ready for transformation. Use 1-2 μl of annealing reaction for each transformation, following a standard...stability to hold the plasmid together through the transformation/replication process. LIC employs long overhangs...association between fragments, allowing for transformation without ligation. Because of its dual polymerase...same principle (homologous to the first G on 3'-5' strand from cut site). For simplicity, only the 5' primer...heating to 75° for 20 minutes. Step 6: Anneal and Transform Mix your treated vector and insert at a molar ...

... tips Bacterial Transformations We walk you through a standard plasmid transformation protocol and offer...tricks to reduce transformation time and increase efficiency Bacterial Transformation Protocol Inoculating...when requesting samples from Addgene Technology Transfer How to Order A step-by-step walkthrough of Addgene's...Addgene's Business Development Manager, discusses his transition from the Scientist Team to Business Team, his...

...practical lab protocols that you can use for a wide range of applications, with videos for select protocols...Ligation Assemble DNA using DNA ligase Bacterial Transformation Introduce DNA into a bacterial strain Watch...Virus Name Description Link to Video General Transfection Introduce plasmid DNA to mammalian cells Lentivirus...Produce lentivirus with a polyethyenimine (PEI) transfection protocol Fluorescence Titering Assay for Lentivirus...preparation Antibodies Name Description Link to Video Transfection Introduce plasmid DNA to mammalian cells to ...