... mL of DMEM complete containing the appropriate antibiotic. This is the beginning of the selection process... antibiotic selection may be a vital step for obtaining a population of cells that have taken up the lentiviral... (am): Remove media, replace with fresh media containing selection reagent Day 3–14: Change media as needed...Hazardous waste container Reagents DMEM high glucose, Corning 10-013-CV L-alanyl-L-glutamine (or alternative ...alternative stable glutamine such as glutaGRO, Corning 25-015-CI) Heat-inactivated FBS Polybrene (10 mg/mL), Millipore...-G 1X PBS pH 7.4 without calcium or magnesium, Corning 21-040-CV (cations can affect the attachment of...Neptune 3745.X 6-well tissue culture treated dish, Corning 3516 15 mL conical tubes, VWR 21008-216 Lentivirus...

... with one well remaining as the untransduced control). Count the cells in the remaining well, a cell count...preparations that carry a fluorescence marker. Determining the titer of your lentiviral vector allows you...still be used in downstream applications. Safety Warnings Lentivirus is generally considered biosafety level...fluorescence microscope Reagents DMEM high glucose, Corning 10-013-CV L-alanyl-L-glutamine (or alternative ...alternative stable glutamine such as glutaGRO, Corning 25-015-CI) Heat-inactivated FBS Polybrene (10mg/mL), Millipore...-G 1X PBS pH 7.4 without calcium or magnesium, Corning 21-040-CV (cations can affect the attachment of...Neptune 3745.X 6-well tissue culture treated dish, Corning 3516 15 mL conical tubes, VWR 21008-216 Lentivirus...

...steps for lysing cells, determining total protein concentration in the lysate, running a precast SDS-PAGE ...Close the clamp. Prepare 1X running buffer as follows: Dilute 25 mL of 20X running buffer to 500 mL with deionized...protein loading buffer Precast SDS-PAGE gel SDS-PAGE running buffer Prestained protein ladder Ethanol iBlot ...the chamber of the SDS-PAGE gel tank with the 1X running buffer. Load the samples into the gel. Load 5–10...gel. Increase the voltage to 150 V and continue running the gel until you have obtained the desired separation...Refer to the manufacturer's information before running your experiment. Remove the membrane from the transfer...

...glutaGRO, Corning 25-015-CI) Heat-inactivated FBS 1X PBS pH 7.4 without calcium or magnesium, Corning 21-040...well tissue culture treated dish, Corning 3516 96-well plate, Corning 3596 0.45 μm polyethersulfone filter... Protocols Cloning by Limiting Dilution Isolating a Monoclonal Cell...new window) or isolating individual cells with cloning cylinders. Workflow Timeline This protocol begins...Polyclonal stable cell pool DMEM high glucose, Corning 10-013-CV L-alanyl-L-glutamine (or alternative ...strainer mesh (optional) 10 cm tissue culture dish, Corning 430167 (optional) Reagent Preparation DMEM Complete... will help you focus the microscope later when scanning the plate for individual cells, which can be difficult...

...175 flask, Corning 430825, 175 cm 2 Cellstack 5, Corning 3319, 3180 cm 2 Cellstack 2, Corning 3269, 1272...high glucose, Corning 10-013-CV DMEM, low glucose (1 g/L) glucose, sodium pyruvate, Corning 10-014-CV (optional...(optional) 7.5% sodium bicarbonate, Corning 25-035-CI (optional) 1 M HEPES, HyClone SH30237.01 (optional...alternative stable glutamine such as glutaGRO, Corning 25-015-CI) Opti-MEM, Thermo Fisher 31985-070 0.05%...12605010) 1X PBS pH 7.4 without calcium or magnesium, Corning 21-040-CV (cations can affect the attachment of... bottle. Add each plasmid DNA into the bottle containing the Opti-MEM. Mix well. Add 4 mL of PEI (1:2 ...the OptiMEM + DNA + PEI solution to the bottle containing 774 mL of DMEM + 2% HI-FBS. Mix well. Take the...

...Considerations For antibody-expressing plasmids containing the SV40 origin of replication use a HEK293 line...large T antigen. For antibody-expressing plasmids containing the Epstein-Barr virus origin of replication ...expressing Epstein Barr nuclear antigen (EBNA). Safety Warnings HEK293 cells are considered biosafety level 2....Pluronic F-18, Thermo Fisher 24040032 Glutagro, Corning 25-015-CI BalanCD HEK293 Media, Irvine Scientific... Sigma 10791 Leupeptin, Millipore Sigma L2884 Aprotinin saline solution, Millipore Sigma A6279 Before ...Leupeptin 50 mg Antipain 250 mg Benzamidine 25 mL Aprotinin saline solution (2 mg/mL) Mix well and sterilize...µL of cell suspension into the microfuge tube containing the trypan blue. Pipette 10 times to mix. Load...

...it to the running buffer when you run the gel. If you do not add EtBr to the gel and running buffer, you...determine the approximate length of a DNA fragment by running it on an agarose gel alongside a DNA ladder (a ...the gel is poured into it. Loading Samples and Running an Agarose Gel: Add loading buffer to each of your... solution and rinse with water to even out the staining after the gel has been run, just as you would ...gel into a container filled with 100 mL of TAE running buffer and 5 μL of EtBr, place on a rocker for ...conducting certain procedures, such as molecular cloning, you will need to purify the DNA away from the ...resolution (crispness) of your DNA bands include: a) running the gel at a lower voltage for a longer period ...

...like... Plasmids 101 Blog Posts Mol Bio Protocols Cloning Protocols Introduction Plasmids can carry one or...allows researchers to easily isolate bacteria containing that plasmid from bacteria that do not contain...maximum of ~100 colonies Pro-Tip If you will be screening a large number of colonies, we recommend using...potentially more effective at selecting only bacteria containing the plasmids of interest (for example, fewer ...swirl to form a medium/agar colloid. Cover the opening of the bottle with its cap or aluminum foil (but...for your bottle of molten gel mix, a tube rack containing the appropriate antibiotics, and a section for...sufficient water to submerge ~75% of the bottle containing your molten gel mixture. Pro-Tip You will cool...

...well with 200 µL 1X MOPS running buffer. Fill the chamber with 1X MOPS running buffer. Make sure that the...standard antibody of known concentration. After staining with Coomassie blue, protein band intensities ...thick, 10-well, Invitrogen NP0321BOX 20X MOPS SDS running buffer, Invitrogen NP0001 4X NuPage sample buffer...Pipettes, 50 mL, VWR 89130-902 Gel loading tips, Corning 4853 Microcentrifuge tubes, Neptune 3745.X PBS,... pH 7.4, VWR 45000-446 250 mL sterile bottles, Corning 430281 Deionized water IgG isotype standard 2.5... at 150 V for 1 h . Pro-Tip If the samples are running unevenly and the dye front looks like a “smile”...part of the gel where dye is visible. Section 2: Staining the Gel Place the gel in a plastic tray with 100...

... Protocols Gibson Cloning Gibson Assembly Cloning You may also like... Cloning Methods...Assembly Cloning! Protocols...biologists now use this method extensively. Why Gibson Cloning? No need for specific restriction sites. Join almost...order primers (see figure to the right). When designing your plasmid, think about what DNA segments you...and enrich for correctly assembled plasmids by designing primers to split an antibiotic resistance gene...fragments together using oligos When you need intervening sequence between two PCR products, one method...

...cells for 18 h, or until the following morning. The following morning, carefully aspirate the media. Replace... Magnetic Stir Bar Reagents DMEM high glucose, Corning 10-013-CV L-alanyl-L-glutamine (or alternative ...alternative stable glutamine such as glutaGRO, Corning 25-015-CI) Low serum medium such as Opti-MEM or Opti-Pro ...FBS 1x PBS pH 7.4 without calcium or magnesium, Corning 21-040-CV (cations can affect the attachment of...tubes, Neptune 3745.X 10 cm tissue culture dish, Corning 430167 15 mL conical tubes, VWR 21008-216 Hydrochloric...health of the packaging cell line is critical for obtaining high viral titer. 293T cells should be split 3...incubation, add the DNA:PEI-Max mix to the conical containing the media and chloroquine and mix well. Gently...

...Tips on designing primers for PCR... Protocols Designing Primers How to Design a Primer You may also like...like... Polymerase Chain Reaction Plasmid Cloning by PCR Agarose Gel Electrophoresis Primer Design for ...PCR Oligonucleotide primers are necessary when running a PCR reaction. One needs to design primers that...region of DNA. They are synthesized chemically by joining nucleotides together. One must selectively block...DNA strand so elongation can proceed. Usually a guanine or cytosine is used at the 3’ end, and the 5’ end... and disrupts the amplification process. When designing, if unsure about what nucleotide to put at a certain...

... to support reproducibility in science. Safety Warnings Sodium azide is toxic. When handling, use proper... laboratory goggles, and gloves. Sodium azide containing waste should be properly disposed of following... Sigma 10791 Leupeptin, Millipore Sigma L2884 Aprotinin, Millipore Sigma A6279 Zeba Spin Desalting Columns... antipain 250 mg benzamidine 25 mL of 2 mg/mL aprotinin Mix well and sterilize through a 0.2 µm PES filter...Equilibrate filtered tissue culture supernatant containing the recombinant antibody to room temperature ...column and collect 500 µL fractions into the tubes containing 50 µL 1 M Trizma hydrochloride pH 9.0. Cap the...sample to the reservoir of the column. Fill the remaining space in the column with PBS and pipette several...

...cells for 18 h, or until the following morning. The following morning, carefully aspirate the media. Replace...Hazardous waste container Reagents DMEM high glucose, Corning 10-013-CV L-alanyl-L-glutamine (or alternative ...alternative stable glutamine such as glutaGRO, Corning 25-015-CI) Heat-inactivated FBS Low serum medium such as ...tubes, Neptune 3745.X 10 cm tissue culture dish, Corning 430167 Hydrochloric acid Sodium hydroxide 0.22 ...Start The health of the cell line is critical for obtaining high levels of virus. HEK293T cells should be ...aspirate media, add 10 mL fresh DMEM complete containing 25 µM chloroquine diphosphate and incubate ~5...

...Place 100 mL sterile LB at 4 ℃. Day 2 (morning) Before beginning, prechill at least four conical tubes ...plasmids in a single sample. They are often used for screening, barcoding, or other high throughput multiplexed...between their LTRs resulting in a smaller plasmid containing only the elements required for bacterial propagation...) Cells should be harvested first thing in the morning to ensure that outgrowth and competition are limited...from cuvette and add to 14 mL vented Falcon Tube containing 3 mL SOC. Repeat for each of the 25 µl aliquots...not pipette repeatedly or mix when removing SOC containing transformed DNA-Endura from cuvette. Shake four...for dispensing cold LB and one for removing LB containing bacteria from plate). Pro-Tip Scraped bacteria...

...conveniently plate and select transformed cells containing plasmids differing in their resistance genes,...need to prepare separate batches of antibiotic-containing agar. This protocol will focus specifically on...antibiotic) E. coli transformed with a plasmid containing the carbenicillin (amp) resistance gene (or other...potentially more effective at selecting only bacteria containing the plasmids of interest (for example, fewer ...more expensive. With a 6 cm diameter petri plate containing solidified LB-agar, pipette 150 µL of carbenicillin... a very small visible volume of pooled liquid remaining on the surface). We use the micropipette tip itself...

...commonly used in molecular cloning techniques, such as PCR or restriction cloning . It is also used to quickly...typically involves ∼500 ng of DNA, while molecular cloning often requires 1 µg of DNA. The total reaction ...sufficient. For digests with >1 µg of DNA used for cloning, it is recommended that you digest for at least...finding an enzyme that cuts your vector's multiple cloning site (MCS), but does not cut your insert, you could...prevent re-circularization of the vector if you are cloning in an insert. CIP (calf alkaline phosphatase) or...

...Bacterial Transformation You may also like... Plasmid Cloning by Restriction Enzyme Digest Restriction Digest...approximately 20-30 mins). Remove agar plates (containing the appropriate antibiotic ) from storage at ...be able to grow once plated on the antibiotic containing agar plate. This step is not critical for Ampicillin...the transformation onto a 10 cm LB agar plate containing the appropriate antibiotic. Pro-Tips We recommend...of the plasmid) you can save a lot of time by shortening or skipping many steps and will still get enough...Check that you are plating on an LB Agar plate containing the correct antibiotic. The resistance gene on...

...requires free nucleotides [dNTPs; adenine (A), cytosine (C), guanine (G), thymine (T)] in an equal molar...our protocol on plasmid cloning by PCR to learn how to design primers for cloning purposes. What do I do...Chain Reaction (PCR) You may also like... Plasmid Cloning by PCR Restriction Digest of Plasmid DNA DNA Ligation...DNA synthesis. Sterile dH 2 O: Used to fill the remaining 50 μL of the reaction mix. Its solvent and buffer...

...technique for molecular cloning, such as PCR - or restriction enzyme -based cloning. Protocol: Gel Purification... can be achieved by using a wider gel comb and running the gel at a lower voltage. You will want to have...resolution (crispness) of your DNA bands include: a) running the gel at a lower voltage for a longer period ...bands? If you have similarly sized bands that are running too close together you can adjust the gel percentage...