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...molecular biology techniques. Topics include: Genome Engineering Fluorescent Proteins CRISPR Viral Vectors Plasmid...

... Protocols Recovering Plasmid DNA from Bacterial Culture Recovering Plasmid DNA from Bacterial...with your plasmid Resuspension buffer Denaturing solution Renaturing solution 2 mg/mL RNase A TE or water-saturated...solution. Add a renaturing solution to the denatured bacteria. Note: This step brings the pH back down...that is compatible with the next solution. Add a denaturing solution to the resuspended bacteria. Note: This...mM EDTA Store Solution I at 4°C Solution II - Denaturing Solution 0.2 N NaOH 1.0% SDS Store Solution II... II at room temperature Solution III - Renaturing Solution (Potassium Acetate) 120 mL 5M Potassium acetate...supernatant into a new tube by pipetting or carefully pouring. Optional: Add 5 μL of 2 mg/mL RNase A to the supernatant...

...a variety of applications. Sharing speeds science. We believe that sharing the full details of our protocols...mL luer-lock syringe, BD BD302832 0.2 µm luer-lock filter, VWR 431229 0.22 µm PES filtering system, 1000...1000 mL, VWR 431098 0.45 µm PES filtering system, 500 mL, VWR 430770 Trypan Blue, Thermo Fisher T10282 ... reagents Store at 4 °C until use. We suggest preparing fresh solutions after one month. BCD Feed 500 ... Glutagro Store at 4 °C until use. We suggest preparing fresh solutions after one month. 1000X protease...mL) Mix well and sterilize through a 0.2 µm PES syringe filter. Aliquot and freeze upright at -20 °C. Procedure...Section 3: BCD Feed and valproic acid supplementation During the 24–144 h post-transfection, supplement the ...

...to a refrigerated centrifuge, you can carefully bring a centrifuge into a cold room for centrifugation...centrifugation. Once you’re done using the centrifuge, bring this equipment back to room temperature, as prolonged...Add the correct amount of 7.5 M LiCl solution to bring the concentration of LiCl in the extracted aqueous...sample, consider making smaller aliquots of it and storing those in -80°C. Option #2 - TRIzol® Protocol Homogenize...

...siRNA selection tool to determine a set of top-scoring targets for your gene. For example, the Whitehead...alleviate concerns about off-target effects. B.2 Ordering Oligos Compatible with pLKO.1 To generate oligos... 5 μL 10x NEB buffer 1 1 μL AgeI add ddH 2 O to bring to 50 μL final volume Incubate at 37°C for 2 hours...ligase buffer 1 μL NEB T4 DNA ligase add ddH 2 O to bring to 20 μL final volume Incubate at 16°C for 4-20 ...for EcoRI 0.8 μL EcoRI 0.8 μL NcoI add ddH 2 O to bring to 20 μL final volume Incubate at 37°C for 1-2 hours...HEK-293T cells that were inadvertently collected during harvesting. TIP: In lieu of centrifugation, you...based on your experiment Detailed protocols for preparing polybrene, protamine sulfate, and puromycin are...

...step-by-step walkthrough of Addgene's ordering process Ordering Page MTA FAQs Answers to frequently asked...Plates Create plates to culture bacteria in the lab Pouring LB Agar Plates Protocol Streaking Bacteria on Plates...enzymes Diagnostic Restriction Digest Protocol Storing and Handling Addgene Plasmids What to do after ...receive your plasmids from Addgene Instructions for Storing and Handling Plasmids Genomic Deletions with CRISPR...Australia, and advice for current graduate students considering their future careers. Eric J. Perkins, PhD In...

...~7.4. Autoclave or sterile filter. Pro-Tip Stirring during the cooling period is recommended or the solution...glucose, lower FBS media causes less filter clogging during harvesting. The addition of sorbitol has been shown...PEI powder into 100 mL of deionized water. While stirring, slowly add hydrochloric acid until the solution... challenging due to the high viscosity of PEG. Stirring under medium heat will promote faster dissolution...overgrow your cells. Pass the cells twice a week during the maintenance phase and do not allow cells to... °C for 1 h, then keep at 4 °C for 3 h without stirring to allow full precipitation. Precipitation of ...

... of reagents and a basic heating and cooling (denaturing and annealing) cycle. The process became automated...in an appropriate buffer, a series of heating (denaturing) and cooling (annealing) steps allow the Taq ...template strand to the point where the strands start denaturing and the hydrogen bonds are broken between the... tops to the PCR tubes, gently tap each tube to bring all the liquid to the bottom before placing it in...sequence is GC-rich, increase the time of the denaturing. Your 5’ and 3’ primers should be designed to... that can withstand radical temperature changes during a typical PCR. The DNA polymerase has an optimum...

...pipette shows the volume adjustment ring right below the plunger. This ring changes the pipette volume. Below... volume for the the pipette, and the tip ejector ring at the bottom which pushes the pipette tip off of...tip is so that the same pipette can be used for measuring different samples without cross contamination ...your fingers to avoid contaminating the tip or puncturing your glove. Ensure that the tip is properly set...

... particular sample. For additional tips on AAV titering using ddPCR, read this blog post on ddPCR for ...all the way, just ensure the samples are covered. Bring the PCR tubes to the BSC used for dilutions. Without...Lightly cap the PCR tubes. Generate the Droplets Bring the PCR tubes to the droplet generation BSC. Without...samples on ice or pre-chilled 96-well freezer blocks during use. In this protocol, a dilution series is prepared...dilutions and titrations For additional tips on AAV titering using ddPCR, read our blog post. Last reviewed...

...like... Inoculating a Liquid Bacterial Culture Recovering Plasmid DNA from Bacterial Culture Restriction...Restriction Digest of Plasmid DNA Background Information During several different stages of molecular cloning, ...specific wavelength that they maximally absorb. By measuring the absorbance of a liquid you can accurately ...specific to your lab. Spectrophotometer Tips Before measuring any samples, be sure to ‘blank’ the spectrophotometer...

...as possible due to the inherent possibility of altering the composition of the library. Bottlenecks, fitness...representation of individual plasmids in the pooled library during amplification. This protocol is designed to be ...pulling motion Take care not to split or gouge agar during the scraping process. Add each scrape into a 50...efficiency! Ensure that no arcing is taking place during electroporation. Arcing would manifest as a loud... often accompanied by some light in the cuvette during the electroporation step. I got a heavy recombined...

... basic molecular biology, plasmid cloning, and titering and testing your viral preparations. ...targeting human and mouse genes. Virus Protocols for titering and testing your virus preparations. Name Description...polyethyenimine (PEI) transfection protocol Fluorescence Titering Assay for Lentivirus Quantify virus using fluorescence...fluorescence measurements Colony Formation Titering Assay for Lentivirus Quantify virus by counting antibiotic...

...antibiotic to the correct concentration ( see Preparing Antibiotics ). Note: If you intend to do a miniprep...without growth (top) and with growth (bottom). Preparing Antibiotics Create a stock solution of your antibiotic...Addgene recommends making 1000X stock solutions and storing aliquots at -20 °C. To use, dilute your antibiotic...Glycerol Stocks for Long-term Storage of Plasmids Recovering Plasmid DNA from Bacterial Culture All Addgene...

...QuickSeal tube spacers 16 ga needle 18 ga needle 10 mL syringe 18 ga blunt edge needles, Hamilton 1X PBS pH 7.4...QuickSeal tube in the order below using a 10 mL syringe and a blunt edge 18 ga Hamilton needle, taking ...interface with an 18 ga needle attached to a 10 mL syringe. The bevel of the needle should be up, facing the... If the concentrate volume is less than 500 µL, bring up the volume with formulation buffer. Use a P1000...

...loading buffer, New England Biolabs B7022S Procedure Pouring a Standard 1% Agarose Gel: Measure 1 g of agarose...the solution heats up.). Caution HOT! Be careful stirring, eruptive boiling can occur. Pro-Tip It is a good...on the sample to prevent bubbles or buffer from entering the tip. Place the very top of the tip of the ...Note: When using UV light, protect your skin by wearing safety goggles or a face shield, gloves and a lab...

...Protein A or Protein G columns. Sharing speeds science. We believe that sharing the full details of our protocols...89039-656 Microcentrifuge tubes, VWR 89126-724 Filtering system 0.2 µm PES, VWR 431098 Sodium phosphate...filtration unit, 500 mL, rapid-flow, VWR 73520-984 Syringes, 30 mL, VWR BD302832 Filter, 0.2 µm (luer-lock...

...Making LB Agar Plates Bacterial Transformation Recovering Plasmid DNA from Bacterial Culture Introduction... select transformed cells containing plasmids differing in their resistance genes, as one does not need... give the antibiotic time to more fully absorb. During the incubation, transform DH5α E. coli cells by...

...Restriction Digest of Plasmid DNA You may also like... Recovering Plasmid DNA from Bacterial Culture Purifying ...enzyme digestion takes advantage of naturally occurring enzymes that cleave DNA at specific sequences....Buffer 3 µL 10x BSA (if recommended) x µL dH 2 O (to bring total volume to 30µL) The amount of restriction ...

...hydroxide 0.22 μm polyethersulfone (PES) filter Syringes for filtering Reagent Preparation DMEM Complete: 10% ...of powder into 100 mL of deionized water. While stirring, slowly add hydrochloric acid until the solution...