...for transfecting mammalian cells using linear polyethylenimine. Transfections allow for transient expression...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...adapted for different cell lines and different transfection reagents. Last Upload: November 7, 2023 Workflow...

...Introduction Transformation is the process by which foreign DNA is introduced into a cell. Transformation of bacteria...modifications to create bacterial strains that can be more easily transformed and that will help to maintain...prepared for optimal transformation efficiencies upon thawing. For the highest transformation efficiency, we...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... Protocols Bacterial Transformation Bacterial...

... Protocols ddPCR Titration of AAV Vectors ddPCR Titration of AAV Vectors You...for purified AAV titration. Protocols...You may also like... AAV Titration by qPCR AAV Viral Service Viral Vector Guides Virus Blog Posts Molecular...samples. ITR Master Mix Volume 9X Volume Final Concentration 2X ddPCR Supermix for Probes, no dUTP 10 µL ...the DG8 gasket, making sure that it is secure. Transfer the cartridge holder to the droplet generator....while slowly and gently aspirating the droplets. Transfer the droplets to a prechilled PCR plate. *Pro-Tip... 98 10 2 1 Hold 4 ∞ 2 1 After PCR is complete, transfer the plate to the Droplet Reader. Open the QuantaSoft...

...Colony Formation Titration Assay Lentivirus Fluorescence Titration Assay AAV Titration by qPCR Addgene ... Protocols ddPCR Titration of Lentivirus Vectors ddPCR Titration of Lentivirus...droplets (blue/green). The concentration of RRE positive droplets in the untransduced control should be close...serially diluted and used to transduce HEK293T cells. Genomic DNA is extracted from the target cells and ...+06 7 Untransduced 1 6.4 17940 0.0007134894091 1.43E+02 Table: Example dilutions and titrations Reference...lentivirus vector titration. Protocols...titer range of 1E+05–1E+09 TU/mL, where TU is transducing units and generally represents the number of ...

... 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...

... Protocols RNA Extraction Without a Kit RNA Extraction Without a Kit...effectively lyse open the cells. Extract RNA from the homogenized sample(s).Transfer tissue/cell lysate to a 4....5 M LiCl solution to bring the concentration of LiCl in the extracted aqueous layer to 2.5 M. Incubate...perform RNA extraction without a kit. Protocols...Reference Introduction This protocol describes how to extract RNA from tissues or cells without a kit. The general...include homogenization/lysis of cells or tissues, extraction of RNA, precipitation, and resuspension. This...

...Iodixanol Gradient Ultracentrifugation AAV Purification by Iodixanol Gradient Ultracentrifugation You may also...medicine, iodixanol can be used as an intravenous isomolar contrast agent. In research, iodixanol is used...the column. We recommend concentrating to a minimum of 500 µL. If the concentrate volume is less than 500...Follow this protocol using Iodixanol gradient ultracentrifugation to get pure adeno-associated virus (AAV) ...Timeline Day 1: Purify Day 2: Buffer exchange and concentration Note : Both steps could be completed in one ...one (long) day. Equipment Ultracentrifuge T70i rotor or equivalent Reagents Opti-Prep (60% Iodixanol) QuickSeal... pooled for dialysis. Pool clean fractions. Concentration and buffer exchange: Stocks of Pluronic F68:...

... Protocols AAV Titration by qPCR Using SYBR Green Technology AAV Titration by qPCR...AAV Titration protocol. Protocols... Posts Mol Bio Protocols Introduction The AAV Titration protocol can be used to determine the number of...Size in bp of Addgene plasmid #59462 : 6208 bp Concentration of Addgene plasmid #59462 : 1.07 μg/μL Molecular...again but store at 4°C and use within 1 week. Keep track of the Ct value for each standard over time. They...over from the production process (DNase does not penetrate the virion). 5μL sample + 39μL H 2 O + 5μL 10x...sample (do not vortex) Incubate 30 minutes at 37°C Transfer to ice ** Critical: do NOT treat your plasmid ...

...BSA standard concentration versus absorbance. Extrapolate the total protein concentration of the sample...protocol uses a dry transfer device but can be adapted for wet and semi-dry transfer approaches. Last Update...blocking buffer and optimal antibody concentrations. Consider titrating your antibody to determine the optimal...protein concentration and prepare the lysate for SDS-PAGE Determine the protein concentration using a ...well. Assemble the transfer sandwich as follows: Unseal the iBlot 2 PVDF Mini transfer stack. Set the Top...iBlot 2 Gel Transfer Device. Wet the pre-run gel in deionized water and place it on the transfer membrane... parameters are correct. Transfer for 5–6 min for proteins <30 kDa. Transfer for 8–10 min for proteins...

...-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...the final concentration of polybrene in each well should be 10 µg/mL. *Pro-Tip* Transducing too many cells... may have 1 copy of the transgene, while others have >1 copy of the transgene in their genome. This can...high levels of the transgene may have reduced growth rates, especially if that transgene is toxic. Eventually...

...Polymerase III transcription for generation of shRNA transcripts. cPPT Central polypurine tract, cPPT, improves...a Qiaquick gel extraction kit. Elute in 30 μL of ddH 2 O. Measure the DNA concentration. C.4 Ligating ...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...Digesting pLKO.1 TRC-Cloning Vector C.4 Ligating and transforming into bacteria D. Screening for Inserts D.1 Recommended...materials F.2 Determining the optimal puromycin concentration F.3 Protocol for lentiviral infection and selection...pLKO.1 can be introduced into cells via direct transfection, or can be converted into lentiviral particles...

...Fisher 21028 Protein G GraviTrap columns, Cytiva 28-9852-55 or rProtein A GraviTrap columns, Cytiva 28-9852...and mix well. Uncap the Protein G Gravitrap or rProtein A Gravitrap columns and gently pour off the ethanol...Buffer Reservoirs to the top of the Gravitrap column. *Pro-Tip* Gravitrap columns have a finite binding capacity...the Gravitrap column to a clamp on a clamp stand. Use scissors to cut the bottom of the Gravitrap column...the Gravitrap columns at the bottom. Add 5 mL of Pierce IgG Elution Buffer to the capped Gravitrap column...exchange and concentration with an Amicon column Apply 15 mL of PBS to the reservoir of a Amicon Ultra-4 30 kDa...sample has reached the desired concentration. If the sample concentration is still too low, repeat the ...

...antibiotic concentrations. Antibiotic Concentrations Antibiotic Recommended Stock Concentration Recommended... low a concentration for selection. Negative Result 2: Neither Strains Grows If neither strain grows, ...tested strain is not supposed to be resistant to the antibiotic, (+) indicates that the tested strain is ...use. Negative Result 1: Both Strains Grow Assuming the appropriate strains were streaked on the appropriate...Your antibiotic is at too high a concentration. One or both of your strains are no longer viable. You can... both strains on plates without any antibiotic. Negative Result 3: Only the Non-resistant Strain Grows...non-resistant strain grows, it’s likely that you’ve used the wrong antibiotic, or confused your strains....

...medium at a reduced antibiotic concentration (e.g., half the concentration that was used for the initial...passing through a 40 µm cell strainer mesh. Count the cell concentration in this homogenized cell solution... 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...

...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...0.72 pmol Transfer Plasmid* 1.64 pmol OptiPro SFM to total volume 500 μL *Plasmid concentrations and ratios...optimized for each transfer plasmid. *Pro-Tip* Endotoxins can inhibit transfection; therefore, plasmid...the conical so that the final concentration in the dish (once transfection mixes are added) will be 25 ...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...

...carbenicillin to a concentration of 1 mg/mL – 4 mg/mL in LB medium. The concentration of antibiotic required...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...optimizing the concentration of carbenicillin, plated over-agar for selection of transformed E. coli . Sample...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 ...

...time as the virus-mediated 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...August 31, 2023 Workflow Timeline Day 0: Seed and Transduce Cells Day 2: Replace media with fresh media containing...brands and lots of FBS can promote or inhibit transfection. Test a variety of brands and lots of FBS to...to the media, as it can affect viability and transduction efficiency. Titer will vary between cell lines... are seeded the day before transduction. Note that in that case, polybrene should only be added at the...the time of viral transduction, and not during the cell seeding step. Procedure Before beginning a colony...

...cells in CS2 Day 2: Seed cells in CS5 Day 3 (am): Transfect cells Day 7 (am): Harvest cells Equipment Class...brands and lots of FBS can promote or inhibit transfection. Test a variety of brands and lots of FBS to...larger volumes 1000 mL 0.45 µm Rapid-Flow PES Filtration Unit, Nalgene 167-0045) *Pro-Tip* Do not use ...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 ... Pass and plate the cells the day before the transfection. Thaw a new vial of cells after 30 passages.... add 2 mL of 0.05% Trypsin/EDTA. Wait ~2 min. Neutralize trypsin by adding 10 mL of DMEM Complete. Pipet...

...phenol-chloroform extract, and precipitate DNA DNA Quantification Measure DNA concentration with a spectrophotometer...using DNA ligase Bacterial Transformation Introduce DNA into a bacterial strain Watch the Video! CRISPR ...features using sequence analysis RNA Extraction Without a Kit Extract RNA from tissues or cells without ...Creating Bacterial Glycerol Stocks Store bacterial strains or plasmids for long term use Watch the Video! ...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...choice AAV Purification by Iodixanol Gradient Ultracentrifugation Purify adeno-associated virus from a preparation...

...step heats the double stranded DNA template strand to the point where the strands start denaturing and ...range to anneal to each of the single stranded DNA template strands. The DNA polymerase is also stable enough...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...