... as an example of LIC experimental design. Search Addgene's collection of empty LIC cloning vectors Protocol...plasmid together through the transformation/replication process. LIC employs long overhangs to form a stable...product is then repaired during the replication cycle. Empty vectors for LIC typically employ type II restriction...Protocols Overview Ligation Independent Cloning (LIC) obviates the need for the time-consuming ligation... the example shown here. The manufacturer of an LIC-specific vector will provide the homologous sequence...Protocol Step 1: Design Your Primers Primer design for LIC is often as simple as using the backbone manufacturer's... protocol . Search Addgene's collection of empty LIC cloning vectors...

... a single reaction Ligation Independent Cloning (LIC) Scarless cloning with Type II restriction enzymes...protocols that you can use for a wide range of applications, with videos for select protocols in the right-hand...protocols are the building blocks for many more complicated procedures. Plasmid Cloning Protocols for constructing...preparations. Antibodies Protocols for common antibody applications. Intro to the Lab Bench Name Description Link...

... all publications arising from the use of this vector. A.2 Map of pLKO.1 pLKO.1 is a replication-incompetent...terminal repeat. f1 ori f1 bacterial origin of replication. Amp R Ampicillin resistance gene for selection...bacterial cells pUC ori pUC bacterial origin of replication. 5’LTR 5’ long terminal repeat. RRE Rev response...it has been diluted. Mix by swirling or gently flicking the tube. Incubate for 5 minutes at room temperature...the walls of the tube. Mix by swirling or gently flicking the tube. f. Incubate for 20-30 minutes at room... on the size of your target plate. TIP: MOI (multiplicity of infection) refers to the number of infecting...Appendix I.1. Sequence of pLKO.1 TRC-Cloning Vector Click here to see the sequence of pLKO.1 TRC-cloning vector...

...) - LiCl selectively precipitates RNA versus DNA or proteins. Add the correct amount of 7.5 M LiCl solution...solution to bring the concentration of LiCl in the extracted aqueous layer to 2.5 M. Incubate at -20°C ... not affect the quality of RNA or downstream applications. *Pro-Tip* To improve yield of RNA, instead ...

...Load 5 μL of each standard in duplicate Load 5 μL of each sample in duplicate. Do not forget to include a...dilution is appropriate). Quality of duplicates: Exclude duplicates from analyses if there is more than...Considerations Always run standards and samples in duplicate at least If possible, include an AAV reference...standard with DNase ** Make 6 serial dilutions, in duplicate, of your standard curve plasmid (2x10 9 stock ...

...The amount of DNA that you cut depends on your application. A diagnostic digest typically involves ∼500 ...volume usually varies from 10-50 µL depending on application and is largely determined by the volume of DNA...manufacturer’s instructions. *Pro-Tip* Depending on the application and the amount of DNA in the reaction, incubation...you will be using the digested DNA for another application (such as a digestion with another enzyme in a...

...bacteria are used as the means for both storing and replicating plasmids. Because of this, nearly all plasmids...expression) carry both a bacterial origin of replication and an antibiotic resistance gene for use as ... microcentrifuge or falcon tube. GENTLY mix by flicking the bottom of the tube with your finger a few ...

...antibody can be purified for use in a variety of applications. Sharing speeds science. We believe that sharing...antibody-expressing plasmids containing the SV40 origin of replication use a HEK293 line stably expressing the SV40 ... containing the Epstein-Barr virus origin of replication use a HEK293 line stable expressing Epstein Barr...

...sufficient quantities of library for experimental applications. Repeated amplifications should be avoided as...required for bacterial propagation (origin of replication and antibiotic selection). This recombination...each 50 µL aliquot of thawed Endura Duos on ice. Flick gently to mix. Electroporate cells (one at a time...

... lentiviral vector allows you to control the multiplicity of infection (MOI) in downstream studies. This... these cells can still be used in downstream applications. Safety Warnings Lentivirus is generally considered...

...lentivirus can be used for a variety of downstream applications such as stable-cell line generation. Last Upload...DNA. Add the diluted PEI dropwise while gently flicking the diluted DNA tube. Incubate the mixture 15-...

...amplify and design primers that will bind to and replicate it. The following image shows the ends of the ...mutation than restriction enzyme based cloning. DNA replication by PCR has error rates that range from roughly...

...BSA standard that range from 0–2000 µg/mL . In duplicate, dilute 10 µL of standard, blank, and lysate samples... nm . Calculate the average absorbance of the duplicate samples on the plate. Subtract the average absorbance...

...mini-prepped DNA onto the pedestal. Close the lid and click measure, be sure to record the concentration and...

...of the UV and with a clean, sterile razor blade, slice the desired DNA fragment from the gel. Note: To ...

.... You can find Addgene's sequencing results by clicking on the "View Sequences" link on the Plasmid Information...

...according to speed and time (and temperature, if applicable) listed in your protocol. *Pro-Tip* Spin speed...

...DNA. Add the diluted PEI dropwise while gently flicking the diluted DNA tube. Incubate the mixture 15-...

... 3:1 ratio is not working or when doing more complicated cloning. While 3:1 will get you in the ballpark...

...Link opens in a new window) . Gibson Assembly® is licensed to New England Biolabs by TelesisBio. Gibson DG...