... extra part, one part has half of the antibiotic gene and the adjacent part has the other half. Any colonies...fully ligated double-stranded DNA molecule. This has proven to be an efficient and effective method for... to improve accuracy and efficiency. Each enzyme has a specific and unique function for the reaction: ...also sell ET SSB (Link opens in a new window) . NEB has other resources, such as a primer design tool (Link...

...continue towards a boil. Keep an eye on it the solution has a tendancy to boil over. Placing saran wrap over ...sit at room temperature for 20-30 mins, until it has completely solidified. Pro-Tip If you are in a hurry... loading and allows you to gauge how far the DNA has migrated; 2) it contains a high percentage of glycerol...with water to even out the staining after the gel has been run, just as you would if you had not added ...water and destain for 5 mins. Using any device that has UV light, visualize your DNA fragments. The fragments...

...weighed out Procedure The balance in the photo above has a capacity of 200 g, as indicated by the number above...determine if this is best. Check that the balance has the correct capacity for the material that you are...

...Multiple Cloning Site (MCS) Adding short tags - ex: HA or Flag Cloning shRNAs Background For the purposes... design). Let's assume that your favorite vector has a relatively limited MCS (BamHI - EcoRI - SalI) and...

...protective measures for each level, as each level has different safety requirements. BSL-1 is designated...is important! Disclaimer: The SOP presented here has been designed by the Addgene nonprofit plasmid repository...

...not require specific PPE, you never know what else has been placed in the centrifuge before you and may ...the centrifuge’s internal lid on the rotor (if it has one) and then close the external lid. Adjust the ...

...the future you can thaw a new tube that you know has only been thawed once before. Always do controls....want to see is that your vector + insert ligation has many more colonies than your vector alone ligation...

...starting this procedure, be sure that the cell pool has been sufficiently selected with the appropriate antibiotic... antibiotic so that every cell in the culture has, in theory, been transduced. There are various schools...regularly for transgene expression. This protocol has been optimized for adherent cells but can be modified...

...). Pro-Tip Add protease inhibitors to 1X if this has not already been done. We typically start with about... NanoDrop Spectrophotometer to see if the sample has reached the desired concentration. If the sample ... NanoDrop Spectrophotometer to see if the sample has reached the desired concentration. Gently transfer...

...clogging during harvesting. The addition of sorbitol has been shown to increase viral titers by up to 1.8-...10 min and then recheck the pH to ensure that it has not drifted. Filter the solution through a 0.22 μm...such as sorbitol and sodium bicarbonate, sorbitol has been found to increase viral titers by up to 1.8-...

... it is possible that the difference between them has affected the accuracy of the NanoDrop. It is a good...

...polyclonal cell population, meaning that the transgene has integrated in different locations in the various ...variety of dilutions and pick the population that has the most desirable level of expression. Over time...

...Extend DNA for 1 minute at 72°C: The Taq polymerase has an optimal temperature around 70-75°C so this step...changes during a typical PCR. The DNA polymerase has an optimum temperature around 70°C and is the molecule...

...final volume of 1 L and recheck pH to ensure that it has not drifted. Filter the solution through a 0.22 µm...incubate for 1 h at room temperature. After the BCD TFX has warmed in the bead bath, transfer the tubes to the...

...the 3’ end, and the 5’ end of the primer usually has stretches of several nucleotides. Also, both of the...

...an impure AAV preparation. The 15% iodixanol step has 1M NaCl to destabilize ionic interactions between... and forth a few times to mix the iodixanol that has settled at the bottom of the column. We recommend...

...is important! Disclaimer: The SOP presented here has been designed by the Addgene nonprofit plasmid repository...

...periodically to determine when the desired color change has occurred. The color will change from yellow to blue...experimental conditions. When the desired color change has occurred, gently remove the plate seal. Using a multichannel...

...vector is the backbone upon which The RNAi Consortium has built a library of shRNAs directed against 15,000...shRNA insert. The original pLKO.1-TRC cloning vector has a 1.9kb stuffer that is released by digestion with... the sequencing conditions if the DNA polymerase has difficulty reading through the secondary structure... in contact with the walls of the tube before it has been diluted. Mix by swirling or gently flicking ...

...provided by your sequencing provider. My sequence has “N”s in it- what does that mean? “N” is the letter...