...Microcentrifuge tubes, Neptune 3745.X 10 cm tissue culture dish, Corning 430167 Hydrochloric acid Sodium hydroxide...solution to mix for 10 min and then recheck the pH to ensure that it has not drifted. Filter the solution through...the best ratio of mass DNA:mass PEI determined. Procedure Seed HEK293T cells at 3.8x10 6 cells per plate...plate in DMEM complete in 10 cm tissue culture plates. Incubate the cells at 37 °C, 5% CO 2 for ~20 h. Gently...gently flicking the diluted DNA tube. Incubate the mixture 15–20 min at RT. Carefully transfer the transfection...

...Deionized water Before Starting Refer to the manufacturer's instructions for additional information specific...antibody for primary antibodies raised in a mouse. Procedure Section 1: Lyse cells Centrifuge 5 x 10 6 cells...of the device. Select the desired method and make sure the parameters are correct. Transfer for 5–6 min... will vary between antibodies. Refer to the manufacturer's information before running your experiment....your antibody to determine the optimal dose. To ensure that your antibody is both functioning as expected...

...amplified should be within 1-10 kB in size. The structure of the primer should be relatively simple and ...and contain no internal secondary structure to avoid internal folding. One also needs to avoid primer-primer...disrupts the amplification process. When designing, if unsure about what nucleotide to put at a certain position...content Start and end with 1-2 G/C pairs Melting temperature (Tm) of 50-60°C Primer pairs should have a Tm...

...add antibiotic to a plate after it's already been poured and solidified....Transformation Recovering Plasmid DNA from Bacterial Culture Introduction This protocol describes methodology...resistance gene (or other antibiotic resistance) Procedure Day 1 Prepare carbenicillin to a concentration...your petri plate. Incubate the plate at room temperature for at least 30 minutes with the lid on to give...

...to add short tags or shRNAs to any vector (the procedure will simply differ in terms of primer design)....be performed enzymatically later. Experimental Procedure Digest and purify vector While waiting for your...bench top) allowing for slow cooling to room temperature (~45 minutes). Method #2 Place mixed oligos in...into your favorite competent bacteria and plate. Be sure to pick multiple colonies for mini-prepping and ...

...Introduction Sequence verification of important plasmid features (such as the gene/insert, fusion proteins, point...sequencing primers, including: Is the primer unique? Make sure the primer only anneals once within the entire construct...mis-called? Open the trace file and use the search feature in the program to locate the incorrect sequence...sequence. Look at the peaks in the area and make sure they are justifiable peaks. For instance, in the trace...

...generally considered biosafety level 2+. Please ensure that you are in compliance with your institution...Microcentrifuge tubes, Neptune 3745.X 6-well tissue culture treated dish, Corning 3516 15 mL conical tubes,... supernatants to multiple freeze-thaw cycles. Procedure Seed 75,000 cells into each well of a 6-well dish... the average of multiple dilutions. Sample Data Figure 1: 293T cells were transduced with a range of dilutions...

...Microcentrifuge tubes, Neptune 3745.X 6-well tissue culture treated dish, Corning 3516 15 mL conical tubes,...transduction, and not during the cell seeding step. Procedure Before beginning a colony formation assay, the... the average of multiple dilutions. Sample Data Figure 1: A549 cells were transduced with the indicated...then stained with 0.1% crystal violet and counted. Figure 2: A549 cells were transduced with the indicated...

...so tight that you cannot perform your duties. Be sure to try out different sizes to know your fit. It ...protocol goes a long way. It is the easiest way to ensure that you’re doing your work in the safest way possible...note that you do so at your own risk; you should ensure that any local guidance is also adhered to. None...

...amounts) Appropriate restriction enzyme (see manufacturer's instructions for proper amount) Approrpriate...Approrpriate restriction digest buffer (see manufacturer's instructions) Gel loading dye Electrophoresis buffer... dig one out of the freezer and you are not 100% sure it is what you are looking for, but you have a map...