...video below to get a quick idea of how the whole process works. Equipment Autoclave Water bath Pipetman ...will grow). However, it is also more expensive. Procedure Measure 37g of pre-mixed LB-agar powder per L ...autoclave tape will darken during the autoclave process if your sample has spent at least 10 min at 121...

... suited for analyzing the gene’s function. The process is shown graphically in the following cartoon, ...TGCTTAGCGGCCGCTCAGTACTTCGAGATATGCCA-3’. Experimental Procedure Run PCR and Ppurify the PCR Product Run PCR to...self-ligating recipient plasmid backbone. Transformation Proceed with the transformation according to the manufacturer...

...completely to the template DNA strand so elongation can proceed. Usually a guanine or cytosine is used at the 3...creates primer dimers and disrupts the amplification process. When designing, if unsure about what nucleotide...

... (with appropriate antibiotic) Bacterial stab Procedure Obtain an LB agar plate with appropriate antibiotic...colonies. Once you have single colonies, you can proceed to Recovering Plasmid DNA or use the individual...

...isolate and purify DNA fragments based on size. The procedure starts with standard agarose gel electrophoresis...concentration of the DNA that you isolated before proceeding to your next intended step with the now gel purified...

...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...

...together through the transformation/replication process. LIC employs long overhangs to form a stable association...free nucleotides from your PCR product before proceeding, as they will interfere with the exonuclease ...

...vector from ligating to itself during the ligation process. If the sticky ends on either side of the vector...ligation can be improved by incubation at 37°C. Proceed with bacterial transformation . Tips and FAQ Do...

...polyethylenimine (PEI) transfection protocol. This procedure can be modified for alternative packaging cell...that are below passage 15 for viral production. Procedure Seed 293T packaging cells at 3.8×10 6 cells per...

...filter. Aliquot and freeze upright at -20 °C. Procedure Section 1: Seeding cells The day prior to transfecting...between 1.5–2 x 10 6 cells/mL with >95% viability to proceed with transfection. Transfect the flask containing...

...of Buffer B and 2 mL of 5 M NaCl to 43 mL PBS Procedure Preparation and loading of the iodixanol gradient...0.001% in PBS: 45 mL PBS + 5 ml B + 200 mM NaCl Procedure Cover the filter membrane with 15 mL of 0.1% Pluronic...

...subcloning), including design and experimental procedures....will save you time in future steps. Experimental Procedure Digest your DNA Set up restriction digests for...

... cooling (denaturing and annealing) cycle. The process became automated with the discovery of a heat resistant...Polymerase (5 units/μL) PCR Machine Agarose Gel Procedure Primer Design and PCR Design Primers. See our ...

... be subjected to multiple freeze-thaw cycles. Procedure Before beginning, determine the optimal dose of...antibiotic. This is the beginning of the selection process, which will begin the selection of a stable cell...

...0.15 μL 15 μL Nuclease Free Water 4.7 μL 470 μL Procedure Prepare a plasmid stock of 2 x 10 9 molecules/...contaminating plasmid DNA carried over from the production process (DNase does not penetrate the virion). 5 μL sample...

... mL of deionized water. Gently invert to mix. Procedure Section 1: SDS-PAGE Add 5 µL of 4X sample buffer... indicates that the samples may not have been processed correctly in the cell (e.g., failure to cleave...

...for different cell types. Before starting this procedure, be sure that the cell pool has been sufficiently...well plate gave rise to 15–20 monoclonal lines. Procedure Generating Conditioned Medium (optional) Seed ...

...Pluronic F-68 4475 µL Molecular Biology Grade Water Procedure Preparation Before handling any viruses, get materials...Seal’ button. After the plate has been sealed, proceed to thermocycling. Thermal Cycling Run the following...

...15 mL DMEM Complete 3 µL of 250 U/µL benzonase Procedure Transducing Cells Thaw the virus on ice. Prepare...Seal’ button. After the plate has been sealed, proceed to thermocycling. Thermal Cycling Run the following...

...found at the Broad Institute's TRC shRNA Design Process . To minimize degradation of off-target mRNAs, ...institution-specific instructions. You must follow safety procedures and work in an environment (e.g. BL2+) suitable...