...protocols from Addgene that can be used for basic molecular biology, plasmid cloning, and titering and testing...designed to help you get started in the lab. Basic Molecular Biology These protocols are the building blocks...weigh laboratory materials on a balance Basic Molecular Biology Name Description (Link opens in a new ...

...Restriction enzyme digestion is commonly used in molecular cloning techniques, such as PCR or restriction...digest typically involves ∼500 ng of DNA, while molecular cloning often requires 1 µg of DNA. The total ... from 10-50 µL depending on application and is largely determined by the volume of DNA to be cut. A typical...will need to use a phosphatase to prevent re-circularization of the vector if you are cloning in an insert...sites . Sometimes enzymes cut sequences which are similar, but not identical, to their recognition sites....website about star activity . If you are digesting a large number of plasmids with the same enzyme(s) (for ...

... your ocular lens. For example, if you observe something using the 10x objective and your ocular lens ...transmission electron microscopes that allow us to see cellular components with science-fiction levels of detail...are magnification (the ability to make an image larger) and resolution (the ability to distinguish between...Lenses Focus Knobs (Fine and Coarse) Nosepiece Arm Ocular Lens (Eyepiece) The route the light follows from...objective lens which magnifies the image, then the ocular lens, where the image is further magnified before...objective magnifies it 10 times, and so on. The ocular lens also provides magnification and the power ...diaphragm and use the coarse focus adjustment knob (the larger of the two focus knobs) until you can see the edges...

...cells for mycoplasma (Link opens in a new window) regularly Do not over or under-grow your cells. Thaw a new...well (0 µL lentivirus, above) are dying. Perform regular media changes and monitor the growth of the cells...selection. It is important to monitor these cells regularly and replace the cell media. Cell death by some...cells in the culture, so it is important to do regular media changes and maintain optimal growth conditions... or removed from the culture, check the cells regularly to confirm transgene expression. As polyclonal... individual wells become confluent, expand into larger vessels. A confluent well of a 6-well dish can ...

... Service Viral Vector Guides Virus Blog Posts Molecular Biology Reference Introduction This protocol can...efficient means to scale-up without having to handle a large number of T-175 flasks. Workflow Timeline Day 0:... (PES) filter system, Nalgene, 565-0010 (or for larger volumes 1000 mL 0.45 µm Rapid-Flow PES Filtration...the amount of each plasmid needed to have a 1:1:1 molar ratio with 2 mg total DNA per CS5 Plasmid Plasmid...determine the total μg/bp we need to achieve a 1:1:1 molar ratio of each plasmid: 2000 μg / 24,961 bp = 0.08.... You are now ready to purify your prep . The clarified supernatant can be kept overnight at 4 °C before...

...equipment that helps bring your materials to a particular temperature, catalyze chemical reactions such...such as restriction digests needed in molecular biology, and thaw cell lines. There are many different types...properly maintain the water bath, empty and clean it regularly. Determine the amount of water needed to fill ...out after your incubation is over. Note: If you regularly use a water bath in the lab, you can leave the...previous uses. However, you will still need to regularly clean out the water bath even if you are using...

...microfuge tubes used in labs, though some larger models can hold larger conical tubes. Depending on the centrifuge...They often spin at similar speeds to their smaller cousins, but can hold much larger containers. Ultracentrifuges...generally floor sized centrifuges that can hold larger containers, but they can also rotate at much higher...

...viruses per cellular genome: Since the cellular gene RPP30 is used as a reference to the cellular genome, ...other targets. Primers and a probe against the cellular ribonuclease P/MRP 30 kDa subunit (RPP30) are ... lower or higher dilutions depending on their particular sample. This protocol was modified from the publication...#71205-3 Polybrene 10 mg/mL, Millipore, TR-1003-G Molecular Biology Grade Water, Hyclone, SH30538.02 ddPCR...

... you will be screening a large number of colonies, we recommend using larger plates. Many labs use 100... plates with colored marker - particular colors correspond to particular antibiotics. Position the flame...good idea of what that volume looks like in your particular plate. For the remainder of the plates, pour ...

...for PCR Based Cloning The basic PCR primers for molecular cloning consist of: Leader Sequence: Extra base...TAAGCAGAATTCATGTGGCATATCTCGAAGTAC-3'. For the Reverse Primer, the design is similar, but we need to use the reverse complement to get... get PCR amplification. We can start similarly, taking the final 18bases of the ORF, including the stop...will need to use a phosphatase to prevent re-circularization of the vector. You should treat your digested...Additionally, if your final product is going to be very large (>10kb) you might want to use electro-competent ...

...high molecular weight proteins and higher percentage acrylamide when immunoblotting low molecular weight...and reagents from other vendors should produce similar outcomes when using these protocols. However, please... ideal lysis buffer will vary depending on the cellular location of the protein of interest. RIPA buffer...buffer will vary depending on the sample type and cellular location of the protein of interest. You may need...

... smaller than the vector) a 3 insert : 1 vector molar ratio will work just fine. We recommend around 100ng...proportionally. 1μL of ligase should be sufficient for larger ligation reactions. Because ligase buffer contains...vector Cut vector + Background due to vector re-circularization - most useful for phosphatase treated vector...vectors, this ratio is really meant to refer to the molarity of DNA ends available for ligation. Simply put...

... like... Cloning Methods Empty Backbone Guide Molecular Biology Protocols Summary In 2009 Dr. Daniel Gibson...effective method for the assembly of plasmids, and molecular biologists now use this method extensively. Why...be best when the DNA fragments are present in equimolar concentrations. The Gibson Assembly master mix...the important regions of your final plasmid, particularly the seams between the assembled parts. Tips ...

... the gel in the first place. Carefully load a molecular weight ladder into the first lane of the gel. ...you are conducting certain procedures, such as molecular cloning, you will need to purify the DNA away ...you get better separation of bands? If you have similarly sized bands that are running too close together..., and a lower percentage gel will help separate larger bands. 10% Rule For each sample you want to load...

...cytosine (C), guanine (G), thymine (T)] in an equal molar ratio. It also requires two unique single stranded...copy the DNA in between the oligo primers. This molecular biology technique creates several micrograms of...Your 5’ and 3’ primers should be designed to have similar melting temperatures (Tm). Set the annealing temperature...frozen. DMSO helps with denaturation of the DNA, particularly for GC-rich templates. What does each ingredient...

...and reagents from other vendors should produce similar outcomes when using these protocols. However, please...blade Plastic tray Gel imaging system ImageJ or similar software Reagents Purified recombinant antibody...should be very little background staining. Pro-Tip A large shift in the electrophoretic mobility of the bands...Analysis Use ImageJ (Link opens in a new window) or a similar photo software to determine the relative intensity...

... Service Viral Vector Guides Virus Blog Posts Molecular Biology Reference Introduction This protocol describes... lower or higher dilutions depending on their particular sample. For additional tips on AAV titering using... bucket 96-well freezer blocks (x 3) Reagents Molecular Biology Grade Water, Hyclone, SH30538.02 GeneAmp...10X Buffer 25 µL of 10% Pluronic F-68 4475 µL Molecular Biology Grade Water Procedure Preparation Before...

...samples. This is a commonly used technique for molecular cloning, such as PCR - or restriction enzyme -...you get better separation of bands? If you have similarly sized bands that are running too close together..., and a lower percentage gel will help separate larger bands. 10% Rule For each sample you want to load...

...result in similar sized bands, thus making this simple digest less informative. This is particularly true ...occurring bacterial endonucleases that recognize a large range of DNA sequences. Given the variety of these...

...usually want to start 2 mL in a falcon tube, but for larger preps you might want to use as much as a liter ...individual plasmid within a single bacterial cell. Large plasmids usually have a low copy number (approximately... other hand, smaller plasmids can be present in large numbers, 50 or more per cell and have a high copy...