...selection curve below. Carbenicillin is used here in place of ampicillin because carbenicillin is more stable,...concentrations of carbenicillin plated over-agar. Control Plate with No Carbenicillin Plate shows a lawn...Plating of Carbenicillin. The above graph displays the stock concentration of Carbenicillin stock used...protocol will focus specifically on selection with carbenicillin; however it can be adapted for any antibiotic...antibiotic High concentration (100 mg/mL, 1000x) carbenicillin stock solution in sterile water (or other antibiotic...coli transformed with a plasmid containing the carbenicillin (amp) resistance gene (or other antibiotic resistance... resistance) Procedure Day 1 Prepare carbenicillin to a concentration of 1 mg/mL – 4 mg/mL in LB medium...

...you through Addgene's online resources. Career Videos Get career insight and advice from real Addgenies...discusses her career in science, the importance of international collaboration, and offers career advice for...how-to screen captures, lab procedure protocols, and career advice videos. Educational...protocols, tips on using Addgene's resources, and career advice. For written lab protocol material, visit...depositing plasmids with Addgene Depositing Page Career Videos (Link opens in a new window) Video Link ..., PhD In this first installment of the Addgene Careers series, we sit down with Addgene Outreach Scientist...Scientist Jessica Welch to discuss her career path, differences in research culture between the US and Australia...

...°C and discard the flow through. Add your sample. Spin at 3500 rpm for 5–8 min at 4 °C, discard the flow...syringe and a blunt edge 18 ga Hamilton needle, taking care to avoid bubbles (Figure 1). 8 mL of 15% iodixanol...40% iodixanol step 3 mL of 60% iodixanol step Carefully add up to 5 mL of clarified supernatant on top...alternatively centrifuge for 2 h at 200,000 x g at 18 °C. Carefully take the QuickSeal tubes out of the rotor and ...elutes from the gradients. In a biosafety cabinet, carefully puncture the QuickSeal tube at the interface of...collected corresponds to the 60% phase and can be discarded. The fractions obtained from the 40% phase contain...the 40% step. Collect up to 5 mL per tube, taking care to avoid collecting the proteinaceous material at...

...Bio-Rad, 1863005 DG8 cartridge, Bio-Rad, 1864008 DG8 gasket, Bio-Rad, 1863009 DG8 cartridge holder, Bio-Rad... step 9 to the NTC tube. Place a DG8 cartridge into the cartridge holder. Using a 2–50 µL multichannel...Cover the cartridge with the DG8 gasket, making sure that it is secure. Transfer the cartridge holder to... centrifuge and place on ice. Wipe down a DG8 cartridge holder with bleach and place in the Biological...reservoir. Using a 20–200 µL multichannel pipette, carefully add the dilution buffer to the dilution plate ...Add 20 µL of the master mix to each PCR tube. Be careful to dispense to the bottom of the tube without collecting...reaction mixtures into the middle wells of the cartridge. Add 800 µL of droplet generation oil to a polystyrene...

... access to a refrigerated centrifuge, you can carefully bring a centrifuge into a cold room for centrifugation...interphase layer, and a top, aqueous layer. Take care to not disturb or collect the interphase layer with...be small, RNase-free Glycogen may be used as a carrier to facilitate RNA precipitation. This does not ...Centrifuge for 20 minutes at 10,000 x g at 4°C and discard the supernatant. There should be a gel-like white...Pro-Tip To prevent overdrying, watch the pellet and carefully remove any residual ethanol wash and add RNase-free... the bottom layer will be a red-pink color. Take care to not disturb or collect the interphase layer with...

...attached to the dish surface and should be handled carefully. Avoid touching the cells when replacing media...pyruvate, Corning 10-014-CV (optional) 7.5% sodium bicarbonate, Corning 25-035-CI (optional) 1 M HEPES, HyClone... 7.5 mL 100X glutaGRO, 7.5 mL of 7.5% Sodium Bicarbonate, 7.5 mL 1 M HEPES to 750 mL DMEM + 1 g/L glucose...stored at 4 °C for up to 2 months. After 2 months, discard the tube and thaw a new working stock. 40% POLYETHYLENE...incubator and pour the media into a waste container. Carefully add the OptiMEM + DNA + PEI solution to the CS5...cells (not poured on them) and can be adjusted by carefully tilting the CS5 back and forth. Pro-Tip To help...other components, such as sorbitol and sodium bicarbonate, sorbitol has been found to increase viral titers...

...Bio-Rad, 1863005 DG8 cartridge, Bio-Rad, 1864008 DG8 gasket, Bio-Rad, 1863009 DG8 cartridge holder, Bio-Rad... Generating the Droplets Place a DG8 cartridge into the cartridge holder. Using a 2–50 µL multichannel...Cover the cartridge with the DG8 gasket, making sure that it is secure. Transfer the cartridge holder to... centrifuge and place on ice. Wipe down a DG8 cartridge holder with bleach and place it in the Biological...Add 16 µL of the master mix to each PCR tube. Be careful to dispense to the bottom of the tube without collecting...reaction mixtures into the middle wells of the cartridge. Add 800 µL of droplet generation oil to a polystyrene...

...in a 50 mL conical tube and dispose after use. Carefully pour the filtered and diluted cell culture supernatant...Resin will appear compacted after centrifugation. Discard the flow through into a waste container. Add 5 ... 2 min to remove PBS. Repeat Steps 30–31 3x , discarding buffer from the collection tube each time. Place...Centrifuge at 1000 x g for 2 min to collect the sample. Discard column after use. Determine antibody concentration...min at RT . Centrifuge at 3100 x g for 8 min . Discard the flow through into a waste container. Add the...times to mix. Centrifuge at 3100 x g for 8 min . Discard the flow through into a waste container. Repeat...additional times to ensure a full buffer exchange. Discard the flow through into a waste container. Fill the...

...dissolved in dH 2 O. *Carbenicillin can be used in place of ampicillin. Carbenicillin is more stable, so ...Protocols Cloning Protocols Introduction Plasmids can carry one or more antibiotic resistance genes, which confer...resistance to a specific antibiotic to the bacteria carrying them. The presence of an antibiotic resistance...that antibiotic - usually conferred by a plasmid carrying the antibiotic resistance gene. The following ...100 mg/mL 100 µg/mL Bleocin 5 mg/mL 5 µg/mL Carbenicillin* 100 mg/mL 100 µg/mL Chloramphenicol 25 mg/mL...

... 4 °C prior to use. Thawed aliquots should be discarded after 1–2 months. 1 mg/mL polyethylenimine, linear...stored at 4 °C for up to 2 months. After 2 months, discard the tube and thaw a new working stock. Considerations...DNA tube. Incubate the mixture 15–20 min at RT. Carefully transfer the transfection mix to your HEK293T ...cells. Add the transfection mix dropwise, being careful not to dislodge the cells. Incubate the cells for...the following morning. The following morning, carefully aspirate the media. Replace the media with 15 ...

...move your microscope around the lab, be sure to carry it with two hands - one hand supporting the base...base and the other holding the arm - or use a cart. Revolve the nosepiece so that the lowest power objective...objective. Take your eyes away from the eyepiece and carefully revolve the nosepiece so that the next objective...will notice that the objectives get larger. Be careful to avoid hitting your sample with the objective...laboratory instrument, you should be sure to take care of your microscope. Bumps, scratches, and dust can...

...the second stop and carefully raise the pipette straight out of the buffer. Carefully load your samples ...occasionally as the solution heats up.). Caution HOT! Be careful stirring, eruptive boiling can occur. Pro-Tip It...not added EtBr to the gel in the first place. Carefully load a molecular weight ladder into the first ...the electrodes from the power source, and then carefully remove the gel from the gel box. Optional: If ...

...Ampicillin VWR: #7177-48-2. Use at 100 μg/mL. Carbenicillin VWR: #80030-956. Use at 100 μg/mL. C.2 Annealing...agar plates containing 100 μg/mL ampicillin or carbenicillin (an ampicillin analog). Due to the long terminal... ligation into LB + 100 μg/mL ampicillin or carbenicillin. Day 2: 2. Spin down the cultures and use a ... to 55°C. Add 1mL of 100mg/mL ampicillin or carbenicillin to obtain a final concentration of 100 μg/mL...distribution of any literature by Addgene is not meant to carry with it, and does not grant any license or rights...distribution of materials by Addgene is not meant to carry with it, and does not grant any license, express...

...Bacterial Culture Background Information Plasmids can carry one or more antibiotic resistance genes, which confer...resistance to a specific antibiotic to the bacteria carrying them. The presence of an antibiotic resistance...isolate individual (clonal) colonies of bacteria carrying a specific plasmid. However, a liquid culture ...Concentration Ampicillin 100 µg/mL Bleocin 5 µg/mL Carbenicillin 100 µg/mL Chloramphenicol 25 µg/mL Coumermycin...

... g for 30 sec. Pour off the supernatant, being careful not to disturb the bacterial pellet. Resuspend ...Add 200 μL of Solution II and invert the tube carefully 5 times to mix the contents. The contents will...the supernatant into a new tube by pipetting or carefully pouring. Optional: Add 5 μL of 2 mg/mL RNase A...Pour out the supernatant in the sink. Pro-Tip Be careful, the pellet is harder to see and less well attached...

...Cap the bottle and invert several times to mix. Carefully remove the plate seal from the 96-well plate and...4 °C . Section 3: Primary antibody incubation Carefully remove the plate seal from the 96-well plate and...°C . Section 4: Secondary antibody incubation Carefully remove the plate seal from the 96-well plate and...or overnight at 4 °C . Section 5: TMB reaction Carefully remove the plate seal from the 96-well plate and...

...stored at 4 °C for up to 2 months. After 2 months, discard the tube and thaw a new working stock. The optimal...the transfection mix onto the 10 cm plate, being careful not to disturb the cells. Incubate the cells for...the following morning. The following morning, carefully aspirate the media. Replace the media with 10 ...

...polymerase and a blend of dTTP/dUTP to minimize carryover contamination. The master mix should also contain...DNase I to eliminate any contaminating plasmid DNA carried over from the production process (DNase does not... 3 Pro-Tip To help stabilize the standards add carrier DNA to a final concentration of 4 ug/mL to each...

...agar. This usually takes 1-2 minutes. Critical Be careful not to rip or shred the agar. Do so by gentle spreading...Pushing motion is better than pulling motion Take care not to split or gouge agar during the scraping process...library even in the face of a recombined fraction but care must be taken to perform adequate NGS based QC to...

...spills produced during the protocol are cleaned up. Discard the weighing boat or weighing paper in the trash...weighed is not biohazardous. If it is biohazardous, discard the weighing boat or paper in the biohazard solid...