...number of unique plasmids in a pooled library can range from a few hundred to millions. Pooled libraries...

...self-ligating recipient plasmid backbone. Transformation Transform your ligation reaction into your bacterial...from your transformation will give you the first indication as to whether your transformation worked. our...Digest of Plasmid DNA DNA Ligation Bacterial Transformation Summary The following technique can be used...competent cells. For most standard cloning, you can transform 1-2μl of your ligation reaction into competent...conduct a positive control to ensure that your transformation worked. You should also verify that you are...

...now ready for transformation. Use 1-2 μl of annealing reaction for each transformation, following a standard...stability to hold the plasmid together through the transformation/replication process. LIC employs long overhangs...association between fragments, allowing for transformation without ligation. Because of its dual polymerase...same principle (homologous to the first G on 3'-5' strand from cut site). For simplicity, only the 5' primer...heating to 75° for 20 minutes. Step 6: Anneal and Transform Mix your treated vector and insert at a molar ...

... tips Bacterial Transformations We walk you through a standard plasmid transformation protocol and offer...tricks to reduce transformation time and increase efficiency Bacterial Transformation Protocol Inoculating...when requesting samples from Addgene Technology Transfer How to Order A step-by-step walkthrough of Addgene's...Addgene's Business Development Manager, discusses his transition from the Scientist Team to Business Team, his...

...practical lab protocols that you can use for a wide range of applications, with videos for select protocols...Ligation Assemble DNA using DNA ligase Bacterial Transformation Introduce DNA into a bacterial strain Watch...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...preparation Antibodies Name Description Link to Video Transfection Introduce plasmid DNA to mammalian cells to ...

...Restriction Digest of Plasmid DNA Bacterial Transformation Background Information The final step in the...the backbone and the complete plasmid can be transformed into bacterial cells for propagation. The majority...recognition sequence, which results in a single stranded overhang on the digested end of the DNA fragment... incubation at 37°C. Proceed with bacterial transformation . Tips and FAQ Do controls When doing ligations...contamination of intact plasmid in ligation or transformation reagents Optimizing the Vector:Insert Ratio...

...Collection Antibody Blog Posts Antibody Guide Transfection for Recombinant Antibodies Recombinant Antibody... Inclusion of this information is solely for transparency intended to support reproducibility in science....125 mg/mL: 10 µL of 0.25 mg/mL with 10 µL PBS Transfer 5 µL of each standard to a microfuge tube. Add... antibody to a concentration of 0.9–1.1 mg/mL. Transfer 5 µL of recombinant antibody to a microfuge tube... menu and select Scatter chart. Change the data range and select the cells where the x-axis is represented...

...material that you are weighing by looking for a weight range on the scale. Make sure that the weight of the material... material you’re weighing out is within this range. If you need less than a gram of material, use an analytical...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...

...of a Gibson Assembly is a fully ligated double-stranded DNA molecule. This has proven to be an efficient...for the reaction: T5 Exonuclease - creates single-strand DNA 3’ overhangs by chewing back from the DNA 5...fragment. Pro-Tip Add Extreme Thermostable Single-Stranded DNA-Binding protein (ET SSB) to the isothermal...You can purchase master mix or make your own. Transform bacteria with the DNA and screen for the correct...

... Inclusion of this information is solely for transparency intended to support reproducibility in science... antibody concentration will vary but generally ranges from 1-10 µg/mL. Add 500 µL of the diluted antibody... antibody concentration will vary but generally ranges from 1-10 µg/mL. Add 500 µL fluorescently-labeled...you know expresses the protein, such as cells transfected with a plasmid to express the protein of interest...

...Bacterial Culture You may also like... Bacterial Transformation Agarose Gel Electrophoresis Agarose Gel DNA...much as several miligrams and at concentrations ranging from 150 ng/μL to several μg/μL. The protocol below...optional) Reagents Overnight culture of bacteria transformed with your plasmid Resuspension buffer Denaturing... a pancreatic ribonuclease that digests single-stranded RNA. Optional: Perform phenol-chloroform extraction...

...sample (do not vortex) Incubate 30 min at 37 °C Transfer to ice ** Critical: do NOT treat your plasmid ...dilutions 5–8 Note: at Addgene, samples typically range from 1 x 10 12 GC/mL to >2 x 10 13 GC/mL and we ... and forth at least 5 times. Seal plate with transparent film. Centrifuge at 3,000 rpm for 2 min to bring...) ~ 1.0, E (efficiency of PCR) ~100% (90%–110% range is acceptable) Baseline removal: all samples will...

...Collection Antibody Blog Posts Antibody Guide Transfection for Recombinant Antibodies Immunocytochemistry... Inclusion of this information is solely for transparency intended to support reproducibility in science...recombinant antibody to room temperature (see Transfection for Recombinant Antibody protocol ). Pro-Tip... has reached the desired concentration. Gently transfer the sample from the reservoir to a LoBind tube...

...outlined in this protocol are based on an AAV titer range of 5E+12–5E+13 GC/mL, where GC is genome copies ...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...

...complementary to template strand). However, primers do not need to correspond to the template strand completely; it...primer corresponds completely to the template DNA strand so elongation can proceed. Usually a guanine or...

... of Plasmids You may also like... Bacterial Transformation Recovering Plasmid DNA from Bacterial Culture... not need to obtain more competent cells and retransform. Bacteria on an LB agar plate can be stored at...frozen bacteria, preventing damage to the cell membranes and keeping the cells alive. A glycerol stock ...

... Inclusion of this information is solely for transparency intended to support reproducibility in science...case, use lower antibody concentrations. The ideal range of the standard curve will vary between targets ...your unknown sample’s absorbance falls above the range of the standard curve you will need to either include...

...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...

...spectrophotometer. A spectrophotometer uses the absorbance/transmission of light through a liquid to determine the concentration...measured under the 260/280 column (A good purity ranges from 1.80-2.00). Repeat for each sample. Notes:...

...Digest of Plasmid DNA DNA Ligation Bacterial Transformation Summary Oligo overlap cloning can be used anytime...the vector with 0.75-6 ng of annealed oligos). Transform 2-3μL into your favorite competent bacteria and...