...2 (am): 18 h post transfection - Remove media, replace with fresh media Day 3 or more (am): Observe fluorescence... (PES) filter Syringes for filtering Reagent Preparation DMEM Complete: 10% v/v FBS and 4 mM L-alanyl-...empirically determined for each new batch of 1 mg/mL PEI prepared. There may be variation between batches of PEI...empirically determined. Once a batch of PEI is prepared, transfect cells with a fluorescent plasmid using...following morning, carefully aspirate the media. Replace the media with 15 mL of DMEM complete. Incubate...

...the full details of our protocols supports reproducibility and accelerates science. Here, we list the ...solely for transparency intended to support reproducibility in science. Last Update: January 20, 2022 Workflow...primary antibodies raised in a mouse. Reagent Preparation Permeabilization buffer: Dilute 20 µL of Triton...100 in 50 mL PBS. 300 nM DAPI working solution: Prepare a 300 µM DAPI stock solution by diluting 2.1 µL...DAPI solution to 100 µL PBS. Protect from light. Prepare a 300 nM DAPI working solution by diluting 5 µL...

...15-25% glycerol. You can prepare the glycerol stock the same time you prepare your plasmid DNA. In the...able to start an overnight culture for plasmid DNA prep the following day. Tips and FAQ The optimal concentration...glycerol stock before you begin your plasmid mini-prep. Try not to freeze/thaw your glycerol stock too ...

...result represents a single experiment. For publishable data, the experiment would need to be repeated to ...their resistance genes, as one does not need to prepare separate batches of antibiotic-containing agar....or other antibiotic resistance) Procedure Day 1 Prepare carbenicillin to a concentration of 1 mg/mL – 4...

...bottom. For the P1000, this represents the ones. For the P200, this represents the first decimal place. ...For the P20, this represents the second decimal place, and for the P2 this represents the third decimal...

... do a mini-prep you will usually want to start 2 mL in a falcon tube, but for larger preps you might want...inoculate bacteria in liquid culture. Protocol Prepare liquid LB. For example, to make 400 mL of LB, weigh...

... but nicked vector product is then repaired during the replication cycle. Empty vectors for LIC typically... plasmid together through the transformation/replication process. LIC employs long overhangs to form a...

...Purification Purify adeno-associated virus from a preparation produced in mammalian cells AAV Purification ... Inoculating a Liquid Bacterial Culture How to prepare and grow bacteria in a liquid medium Inoculating...physical titer of your adeno-associated virus preparation. AAV Titration by qPCR Using SYBR Green Technology...

...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 ...sell competent cells, which come frozen and are prepared for optimal transformation efficiencies upon thawing...

...the new target DNA strand accurately and rapidly. Repeat steps 2-4 25-30 times. Final Extension for 5 minutes...°C below Tm). Extend DNA for 2 minutes at 72°C. Repeat steps 8-10 for 25-30 cycles. Final Extension for...and concentration of PCR product. Master Mix Preparation Multiply the volume of each reagent by the number...

...NanoDrop to measure your samples, place 1-2µL of mini-prepped DNA onto the pedestal. Close the lid and click ...280 column (A good purity ranges from 1.80-2.00). Repeat for each sample. Notes: Keep in mind that despite...

...been designed by the Addgene nonprofit plasmid repository and is being shared outside of Addgene for informational...informational purposes only. If you choose to reuse or repurpose this SOP in another location, please note that...

...many different experimental procedures, such as minipreps and RNA extractions. Plus, centrifuges are useful... are separating solids from a liquid, pellets represent the solids in your sample while the liquid portion...

...been designed by the Addgene nonprofit plasmid repository and is being shared outside of Addgene for informational...informational purposes only. If you choose to reuse or repurpose this SOP in another location, please note that...

...image of that object! Equipment Light Microscope Prepared Slides (or other sample that can fit on a microscope... risk damaging the lens or your sample. You can repeat steps 7 and 8 with progressively higher magnification...

...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...

...together. One must selectively block and unblock repeatedly the reactive groups on a nucleotide when adding...

...plate. Be sure to pick multiple colonies for mini-prepping and verify insert by sequencing. Reference Page...

... antibiotic gene. This trick can also enable replacement of "inverse PCR" reactions with a two-part Gibson...

... μL of EtBr, place on a rocker for 20-30 mins, replace EtBr solution with water and destain for 5 mins...