... as when you have a tube of plasmid DNA and just need to transform bacteria so that you can grow up more...the transformation, so when higher efficiency is needed follow the complete protocol. Thaw the competent...less efficient at taking up larger plasmids. If you need to transform large plasmids, it is a good idea to...induce membrane permeability. To do this you will need to have access to an electroporator and the appropriate...

...concentrations of 0.7% to 2% depending on the size of bands needed to be separated - see FAQs below . Simply adjust...add EtBr to the gel and running buffer, you will need to soak the gel in EtBr solution and then rinse ... procedures, such as molecular cloning, you will need to purify the DNA away from the agarose gel. For...want to load on a gel, make 10% more volume than needed because several microliters can be lost in pipetting...

...alternative cell lines. Workflow Timeline Day 0: Seed and transduce Cells Day 2–3 (am): Remove media, ...containing selection reagent Day 3–14: Change media as needed Day 14–18: Expand and harvest stable cell lines...control well). Perform a "reverse transduction" by seeding 50,000 cells into each well of the 6-well dish....media to make the cell solution in this step. To seed the cells: Prepare a batch of cells as follows: ...

...precast SDS-PAGE gel, and immunoblotting. Sharing speeds science. We believe that sharing the full details.... However, please be aware that the protocol may need to be adjusted to accommodate slight differences...protein is in low abundance in the sample you will need to load a greater amount of total protein. Prepare...cellular location of the protein of interest. You may need to try a variety of lysis buffers to find the best...

...healthy humans. BSL-2 includes all of the precautions needed in BSL-1, however there are additional precautions...shoes Gloves Eye protection and face shields, as needed Guidelines Your lab coat should be buttoned to ...such as goggles and/or face shields can be used as needed. For BSL-2 work always wear glasses/goggles in ...

...a glycerol stock or stab culture of bacteria and need to purify plasmid DNA from it, you will want to ...hours) at 37 °C. Pro-Tip Some plasmids or bacteria need to be grown at 30 °C instead of 37 °C. This is ...colonies. Once you have single colonies, you can proceed to Recovering Plasmid DNA or use the individual...

... Ligation Independent Cloning (LIC) obviates the need for the time-consuming ligation step of traditional...protocol ). After the digest is complete, you will need to separate the linearized vector from the reaction...free nucleotides from your PCR product before proceeding, as they will interfere with the exonuclease ...

... biosafety regulations. Workflow Timeline Day 0: Seed 293T cells Day 1: Transduce cells Day 2 (am): Remove...supernatants to multiple freeze-thaw cycles. Procedure Seed 75,000 cells into each well of a 6-well dish. Prepare...integration event per cell. When the percentage exceeds 40% you risk counting cells with multiple integration...

...broken glass or needles. Sharps containers must be thick walled, impenetrable by a needle, and must be able...cholerae . BSL-2 includes all of the precautions needed in BSL-1, along with additional precautions to ...

...5, 10 or 20μL for storage at -20°C. Whenever you need to set up ligations in the future you can thaw a...ligation can be improved by incubation at 37°C. Proceed with bacterial transformation . Tips and FAQ Do...of DNA no matter how long it is, and therefore we need to adjust the amount of DNA used in a ligation based...

... this method extensively. Why Gibson Cloning? No need for specific restriction sites. Join almost any ... plasmid, think about what DNA segments you will need to join to create your final plasmid. Adjacent segments...Stitching” fragments together using oligos When you need intervening sequence between two PCR products, one...

...that can fit on a microscope stage) Reagents None needed Procedure Set up your microscope by removing any...of view is centered on a region of interest. If needed, you can also use the fine focus knob (the smaller...objective! Once on the higher objective, you will likely need to re-adjust lighting and focus. Increase the power...

...Centrifuge the culture to pellet the bacteria before proceeding with DNA preparation. Pro-Tip If your entire ...DNA to precipitate. Centrifuge solution at high speed (at least 12,000 rpm) for 15-30 min at 4°C. Notes...from the DNA pellet. Centrifuge solution at high speed (at least 12,000 rpm) for 5 min at room temp. Pour...

... GGC TGT TAG AGA GAT AAT TGG A 3’). TIP: You may need to adjust the sequencing conditions if the DNA polymerase...scaled to produce different amounts of virus as needed. Day 1: a. For each plasmid to be transfected, .... Change to fresh puromycin-containing media as needed every few days. h. Assay infected cells. The following...plastic pipettes in place of glass pipettes or needles. Liquid waste should be decontaminated with at ...

...protein content of the sample is determined. Sharing speeds science. We believe that sharing the full details... However, please be aware that your protocol may need to be adjusted to accommodate slight differences...CSV file. Select import. Choose file. Format as needed. Select the Insert tab, then Chart . This will ...

...in your desired bacterial strain and you will not need to obtain more competent cells and retransform. ...want to store bacteria for a longer time, you will need to establish glycerol stocks. The addition of glycerol...

...differing in their resistance genes, as one does not need to prepare separate batches of antibiotic-containing...experiment. For publishable data, the experiment would need to be repeated to account for variability....

...concentration of the DNA that you isolated before proceeding to your next intended step with the now gel purified...want to load on a gel, make 10% more volume than needed because several microliters can be lost in pipetting...

...Summary Oligo overlap cloning can be used anytime you need to add a short stretch of DNA to a plasmid, such...GTATAC AATTAATT CCGCGCGG GTTAAC - 5' = 28 bp We also need to include additional bases to complement the overhangs...

...Before beginning your diagnostic digest, you will need to select a restriction enzyme or enzymes that cut... vector by a single restriction enzyme, you will need to verify that it has be cloned in the correct orientation...