...are commonly referred to as 'competent cells.' Many companies sell competent cells, which come frozen and...instructions that came with your competent cells. *Pro-Tip* Commercial competent cells range significantly in... LB or SOC media Competent cells DNA you'd like to transform Procedure Take competent cells out of -80...efficiency is needed follow the complete protocol. Thaw the competent cells in your hand instead of on...positive control (many companies include a positive control plasmid with their competent cells) to ensure that... To save money, many labs also make their own competent cells. This is a relatively simple procedure and...DNA (usually 10 pg - 100 ng) into 20-50 μL of competent cells in a microcentrifuge or falcon tube. GENTLY...

... Thaw 4 tubes of electrocompetent cells on ice for 15-20 minutes or until completely thawed. Chill a box...obtained. Ensure that electrocompetent cells are being used. Chemically competent cells will not provide... to the inherent possibility of altering the composition of the library. Bottlenecks, fitness differences... in the morning to ensure that outgrowth and competition are limited. Equipment Table top centrifuge BioRad...MicroPulser TM , Bio-Rad 1652100) Reagents 200 µL electrocompetent cells (Default: 4 tubes of Endura Duos, Lucigen...Stbl4 cells or other ultra-high efficiency electrocompetent cells that are suitable for unstable or recombination-prone...recombination-prone DNA. The use of electrocompetent cells is essential to ensure high efficiency uptake...

...layout is complete, select 'Run' to begin the droplet reading. When the droplet reading is complete, export...biosafety level 2+. Please ensure that you are in compliance with your institution’s biosafety regulations...Human, Bio-Rad, 10031244 Reagent Preparation DMEM Complete: 500 mL DMEM, high glucose, pyruvate 55 mL heat...serum 5 mL glutaGRO 50 U/mL benzonase: 15 mL DMEM Complete 3 µL of 250 U/µL benzonase Procedure Transducing...the following dilution series of virus in DMEM complete: Prepare 7 microcentrifuge tubes with the appropriate...Leave one well untransduced (add 150 µL of DMEM complete instead of a viral dilution). Remember, this is...Gently add 1.5 mL of 50 U/mL Benzonase in DMEM complete to each well. Incubate at 37 °C for 30 min. Gently...

...want to use electro-competent cells instead of the more common chemically-competent cells. The number of...sufficient to give specificity and to also be compatible with a standard PCR reaction. Therefore, our...design is similar, but we need to use the reverse complement to get PCR amplification. We can start similarly...the ORF). We now need to generate the reverse-complement of this sequence so that we can successfully ...amplify the ORF. You can generate the reverse-complement using existing software (a quick internet search... one restriction enzyme, or enzymes that have compatible overhangs or no overhangs after digestion, you...according to the manufacturer’s instructions for your competent cells. For most standard cloning, you can transform...

...fragment of interest) into a compatibly digested vector backbone. This is accomplished by covalently connecting...are compatible, meaning that the overhanging base pairs on the vector and insert are complementary, the...is physically attached to the backbone and the complete plasmid can be transformed into bacterial cells... sticky ends on either side of the vector are compatible with each other, the vector is much more likely... will allow you to verify that the vector was completely digested and if phosphatase treated, that the...Interpretation Uncut vector - Checks viability of competent cells and verifies the antibiotic resistance of... 3:1 ratio is not working or when doing more complicated cloning. While 3:1 will get you in the ballpark...

...Lentivirus Preparation Reagent Preparation DMEM Complete: 10% v/v FBS and 4 mM L-alanyl-L-glutamine To ...colony formation assay. Prepare a batch of DMEM complete containing 10 μg/mL polybrene by diluting 20 μL...fold serial dilutions of the lentivirus into DMEM complete containing 10 μg/mL polybrene: Dilution Volume...Lentivirus or Previous Dilution (μL) Volume of DMEM Complete Containing 10 μg/mL Polybrene (μL) Volume of Virus...follows: Dilute 7,000 cells into 9.45 mL of DMEM complete containing 10 μg/mL polybrene. Mix well by pipetting...to 8 h and the media can be replaced with DMEM complete. However, antibiotic-containing media should not...aspirate the media from the cells. Add 1.5 mL of DMEM complete containing the appropriate antibiotic. Incubate...

...of a compound light microscope with the main components labeled: Figure 1: Diagram of a compound light...electron microscopes that allow us to see cellular components with science-fiction levels of detail. You can...common types that you're likely to encounter: the compound light microscope. As the name suggests, light ... make it through the sample because different components of the specimen will refract and reflect the ...) using dyes or stains that add color to some components of the sample but not others. The light that ...that allow you to capture images directly to a computer, but many microscopes do not and you may have ...

...want to use electro-competent cells instead of the more common chemically-competent cells. The number of...version of your insert flanked by restriction sites compatible with the recipient plasmid's MCS. However, you... one restriction enzyme, or enzymes that have compatible overhangs or no overhangs after digestion, you...Follow the manufacturer’s instructions for your competent cells. For most standard cloning, you can transform...transform 1-2μl of your ligation reaction into competent cells such as DH5alpha or TOP10. If using much...getting colonies, you might want to use higher competency cells. Additionally, if your final product is...you used only one enzyme or used enzymes with compatible overhangs you will need to verify the orientation...

...puromycin, blasticidin) Reagent Preparation DMEM Complete: 10% v/v FBS and 4 mM L-alanyl-L-glutamine (or...kills all of the cells. Prepare a batch of DMEM complete + 10 µg/mL polybrene by diluting 20 µL of 10 mg...a range of dilutions of the lentivirus in DMEM complete + 10 µg/mL polybrene. Note, this is just a sample...Dilution Volume of Lentivirus (μL) Volume of DMEM complete + 10 µg/mL polybrene (µL) 0 0 500 1:5 300 200 ...,000 cells into a total volume of 7 mL of DMEM complete + 10 µg/mL polybrene. Mix well by pipetting or...all the media in these wells was made with DMEM complete + 10 µg/mL polybrene, the final concentration ...aspirate the media from the cells. Add 1.5 mL of DMEM complete containing the appropriate antibiotic. This is...

... mM NaCl, 2 mM MgCl2) Reagent Preparation DMEM Complete : 10% v/v FBS and 4 mM L-alanyl-L-glutamine (or...min. Neutralize trypsin by adding 10 mL of DMEM Complete. Pipet back and forth vigorously multiple times...-175 flasks. Adjust volume to 300 mL with DMEM complete media and mix. Seed all cells in 1 CS2. Return...CS2 to help detach the cells, add 200 mL of DMEM complete media, then transfer the cells into a sterile ...sterile bottle. Rinse the CS2 with 100 mL of DMEM complete medium and pool with the cells harvested in the previous... with PEI and allow for the addition of other components, such as sorbitol and sodium bicarbonate, sorbitol...Pipet back and forth to resuspend each pellet completely. Combine resuspended pellets and keep on ice....

... 21-mer targets in your gene B.2 Order oligos compatible with pLKO.1 C. Cloning shRNA oligos into pLKO...vector. A.2 Map of pLKO.1 pLKO.1 is a replication-incompetent lentiviral vector chosen by the TRC for expression...sequence), while the EcoRI site is preserved. For a complete map of pLKO.1 containing the 1.9kb stuffer, visit... nuclear import of the vector’s preintegration complex in the transduced cells. hPGK Human phosphoglycerate...about off-target effects. B.2 Ordering Oligos Compatible with pLKO.1 To generate oligos for cloning into... shRNA sequence flanked by sequences that are compatible with the sticky ends of EcoRI and AgeI. Forward...M0202S T4 DNA ligase buffer NEB #B0202S DH5 alpha competent cells Invitrogen #18258-012 Qiaquick gel extraction...

...works for both of your enzymes. Most companies will have a compatibility chart, such as the double digest ...different enzymes that have compatible sticky ends. See NEB's compatible cohesive ends chart . If you...you cannot find compatible sticky ends, you will need to fill in the overhangs and conduct a blunt end ...Master Mix" consisting of all of the reaction components except for the DNA. Aliquot your DNA into individual...

... 15 mL DMEM complete. Wednesday: Plate 1×10 6 cells in a T75 flask in 15 mL DMEM complete. Friday: Plate...Transfection grade DNAs Reagent Preparation DMEM Complete: 10% v/v FBS and 4 mM L-alanyl-L-glutamine (or...Plate 8×10 5 cells in a T75 flask in 15 mL DMEM complete. Do not add pen-strep to the media. Use cells ...packaging cells at 3.8×10 6 cells per plate in DMEM complete in 10 cm tissue culture plates. Incubate the cells...the media. Replace the media with 10 mL of DMEM complete or OptiPro SFM. Incubate the cells. Virus can ...

...strand completely; it is essential, however, that the 3’ end of the primer corresponds completely to the...reaction. One needs to design primers that are complementary to the template region of DNA. They are synthesized...sequences on the template molecule (must be complementary to template strand). However, primers do not... of each other Primer pairs should not have complementary regions Note: If you will be including a restriction...

...CAATTG ). The bottom oligo will be the reverse compliment so that they can anneal. Top oligo: 5' - CATATG...bp We also need to include additional bases to complement the overhangs generated when digesting the vector...could leave off the 3’ G on each oligo (and the complementary C of the other oligo), but this would destroy...following oligos from your favorite oligo synthesis company: Top oligo: 5' - AATTCCATATGTTAATTAAGGCGCGCCCAATTGG...annealed oligos). Transform 2-3μL into your favorite competent bacteria and plate. Be sure to pick multiple colonies...

...biosafety level 2+. Please ensure that you are in compliance with your institution’s biosafety regulations...Lentivirus preparation Reagent Preparation DMEM Complete: 10% v/v FBS and 4 mM L-alanyl-L-glutamine (or...follows: Dilute 525,000 cells into 14 mL of DMEM complete. Mix well by pipetting or inverting. Aliquot 2... Prepare dilutions of the lentivirus into DMEM complete containing 10 μg/mL polybrene. Note, this protocol...Volume of Lentivirus Stock (μL) Volume of DMEM complete (μL) Volume of 10mg/mL polybrene (μL) 1:10 150...

...DMEM complete containing 25 µM chloroquine diphosphate and incubate ~5 h For 10 mL of DMEM complete, add...Syringes for filtering Reagent Preparation DMEM Complete: 10% v/v FBS and 4 mM L-alanyl-L-glutamine (or...HEK293T cells at 3.8x10 6 cells per plate in DMEM complete in 10 cm tissue culture plates. Incubate the cells...the media. Replace the media with 15 mL of DMEM complete. Incubate the cells 24–48 h before checking for...

...Refrigerator Pipette controller Benchtop centrifuge compatible with 50 mL conical tubes NanoDrop spectrophotometer... 250 mL sterile bottle, VWR 430281 0.45 µm PES complete filtration unit, 500 mL, rapid-flow, VWR 73520...Place a mark on the side of the column where the compacted resin is slanted upward. Place column in centrifuge...centrifugation steps. *Pro-Tip* Resin will appear compacted after centrifugation. Discard the flow through.... Slowly apply the sample to the center of the compact resin bed. *Pro-Tip* For sample volumes <1.5 mL...

...denaturation, annealing, and synthesis. After the PCR is complete, the product can be verified based on size by ...Diluting Primers Most people order primers from a company, which synthesizes and ships them as a lyophilized...essential that the template DNA and primer are complementary for a PCR to work efficiently. Taq Buffer with...Forward and Reverse Primers hybridize and are complementary to the 3’ ends of the sense and anti-sense strands...

...Corning 430167 (optional) Reagent Preparation DMEM Complete: 10% v/v FBS and 4 mM L-alanyl-L-glutamine (or...Each 10 cm dish should be seeded in 10 mL DMEM complete, which will generate enough conditioned medium...conditioned medium, prepare this cell solution in fresh complete medium. This 5 cells/mL solution will be used ...adding 10 µL homogenized cell solution to 990 µL complete medium. Instead of transferring 0.125 µL, now ...