...Molecular Biology Reference How do I process my Addgene pooled library? Introduction Please refer to our pooled... pooled plasmids differ by only a short region of DNA: guide RNAs targeting different genes in an organism's...Picogreen, or Qubit. Each different DNA measurement type can report slightly different concentrations of DNA... Pooled libraries contain tens to millions of different plasmids in a single sample. They are often used...composition of the library. Bottlenecks, fitness differences, and plasmid recombination can all impact the...

... pipette 10–20 times. Use a new pipette tip to transfer 200 µL of the first dilution into the next dilution...min. Resuspend cells in 500 µL DMEM complete and transfer to a microcentrifuge tube. Centrifuge for 5 min...the DG8 gasket, making sure that it is secure. Transfer the cartridge holder to the droplet generator....while slowly and gently pipetting 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...genome: Since the cellular gene RPP30 is used as a reference to the cellular genome, it is assumed that there... cells per well is 300,000 and the virus volume refers to the total volume the cells were transduced, ...

...Lentiviral Packaging plasmid(s), Envelope plasmid, and Transfer plasmid encoding your insert of interest Reagent...and 5 mL of glutaGRO. Store at 4 °C. Pro-Tip Different brands and lots of FBS can promote or inhibit ...1.3 pmol Envelope Plasmid ( pMD2.G ) 0.72 pmol Transfer Plasmid* 1.64 pmol OptiPro SFM to total volume...concentrations and ratios should be optimized for each transfer plasmid. Pro-Tip Endotoxins can inhibit transfection...PEI is 1:3 (1000 μL total per 10 cm dish). Using transfer plasmid pHAGE TRE dCas9-KRAB (total μg of plasmid...gives the highest percentage of GFP positive cells. Refer to the table below for a possible range of ratios...individual harvests are pooled. If pooling harvests, transfer the harvested media to a polypropylene storage...

...Tips Although there are many different types of pipettes that have different mechanisms to disperse liquids...that the same pipette can be used for measuring different samples without cross contamination as long as...performs the same function, the numbers are read differently on each type of pipette. This section will help...Procedure How to Pipette Now that you know the different parts of the pipette, how to select tips, and ...pipette tip. Liquids of various viscosity have different flow rates. The more viscous a liquid, the lower... are pipetting the same amount of liquid into different tubes or into wells of a microplate, you may consider...

...from the final product (no "cloning scars"). A “stuffer” sequence allows for electrophoretic separation...template sequence. 5' and 3' primers will have different leader sequences, but operate on the same principle...at two points by the enzyme BsaI, removing the stuffer sequence and sacB counter-selection marker (see... PCR products with sterile water (instead of TE buffer) to ensure optimal salt concentrations in subsequent... minutes. Volume (μl) Reagent Final Conc. 4 10X Buffer 1X 20-30 Eluted DNA 10-50 ng/μl 1 dGTP (100mM) ...your PCR product before proceeding, as they will interfere with the exonuclease activity of T4 Pol in the...

...restriction enzyme buffers). If you select enzymes that can function in the same buffer, it will save you... to use, you can check out our Empty Backbone Reference . Background Subcloning by restriction digest ... a fusion protein) Ideally, you will find two different restriction enzymes for your subcloning. It is...enzymes will work in the same restriction enzyme buffer (see (Link opens in a new window) New England Biolabs...expression vector and can begin your studies. Reference Page Top Index...

... This approach can be adapted for different cell lines and different transfection reagents. Workflow Timeline...5 mL of 100X glutaGRO. Store at 4 °C. Pro-Tip Different brands and lots of FBS can promote or inhibit ...gives the highest percentage of GFP positive cells. Refer to the table below for a possible range of ratios...Incubate the mixture 15–20 min at RT. Carefully transfer the transfection mix to your HEK293T cells. Add...

...reaction mix) consists of three different enzymes within a single buffer mixture and an optional SSB protein...isothermal reaction. With the activities of three different enzymes, the product of a Gibson Assembly is a...primers to include in the Gibson reaction are different than with normal PCR primers (Gibson et al. , ... than five fragments at a time. Resources and References New England Biolabs (NEB) (Link opens in a new...

... 5mL of 100X glutaGRO. Store at 4 °C. Pro-Tip Different brands and FBS lots can promote or inhibit transfection...efficiency of your transduction, you will see different degrees of cell death upon antibiotic selection...polyclonal populations of resistant cells start proliferating and the individual wells become confluent, expand... meaning that the transgene has integrated in different locations in the various cells in the culture....varying expression levels of the transgene from different cells in the population. Once the polyclonal populations...

...Purification Agarose Gel Purification Molecular Biology Reference Introduction This protocol describes how to extract...ThermoFisher Scientific . Figure 1: A diagram of the different steps in RNA extraction. Before Starting RNA is...cells. Extract RNA from the homogenized sample(s).Transfer tissue/cell lysate to a 4 mL tube. Add the following...the cold room may damage it. Using a pipettor, transfer the top, aqueous phase to a new RNAse-free tube...

...shRNAs to any vector (the procedure will simply differ in terms of primer design). Let's assume that your...oligos The oligos should be resuspended in annealing buffer (10mM Tris, pH7.5-8.0, 50mM NaCL, 1mM EDTA) and...total volume of 50μL - add additional annealing buffer if necessary to get to 50μL. Efficient annealing...mini-prepping and verify insert by sequencing. Reference Page Top Index...

...Introduction For many experiments, you’ll need to make buffers, media, or other solutions. A key part of this ...cross-contamination with other substances. They also help you transfer the material to your tube or container. Pro-Tip...weighed out the correct amount of your reagent, transfer it into your container. Make sure that any spills...the accurate composition of your solutions and buffers. Now that you have your reagents weighed out, you...

...FAQ What is the difference between high copy and low copy plasmids? The copy number refers to the number... one or more antibiotic resistance genes, which confer resistance to a specific antibiotic to the bacteria...350 - 400 rpm to obtain a higher cell density. Reference Page Top Index...

... a standard plasmid transformation protocol and offer some tips and tricks to reduce transformation time...when requesting samples from Addgene Technology Transfer How to Order A step-by-step walkthrough of Addgene's...Scientist Jessica Welch to discuss her career path, differences in research culture between the US and Australia... importance of international collaboration, and offers career advice for scientists looking to expand ...

...can be used to titer lentiviral preparations that confer antibiotic resistance....can be used to titer lentivirus preparations that confer antibiotic resistance. Determining the titer of...5 mL of 100X glutaGRO. Store at 4 °C. Pro-Tip Different brands and lots of FBS can promote or inhibit ... the incubator. Incubate for 48–72 h. Pro-Tip Different cells may be more sensitive to lentivirus or to...

...because different components of the specimen will refract and reflect the light. These differences create... of biological research and are found in many different types of laboratories. These tools allow you to...objective performs essentially the same task but has different magnifying powers. When you look at the side of...

... one or more antibiotic resistance genes, which confer resistance to a specific antibiotic to the bacteria... with resistance to that antibiotic - usually conferred by a plasmid carrying the antibiotic resistance...x (0.220 L) = 8.14 g pre-mixed LB-agar powder. Transfer the LB-agar powder you’ve measured out into an...molten agar from boiling over in the autoclave. Transfer the sterile water (in our case 220 mL) to the ...

...backbone (See Addgene's Sequencing Primers for reference). Many sequencing cores have a list of common ...recommends using Addgene's sequencing results as a reference for primer design. A good sequencing reaction ...reaction with high background: If you obtain a different sequence than expected and wish to contact Addgene...

... potential accidents such as spills. BSL-2 is different because it includes labs that work with agents...cannot perform your duties. Be sure to try out different sizes to know your fit. It is best to not reuse...

... select transformed cells containing plasmids differing in their resistance genes, as one does not need...Sample Data Selection of E.coli on LB-agar using different concentrations of carbenicillin plated over-agar...