...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...

... 15 mL DMEM Complete. Wednesday: Plate 1×10 6 cells in a T75 flask in 15 mL DMEM Complete. Friday: Plate...your insert of interest Reagent Preparation DMEM Complete (also known as D10): 10% v/v FBS and 4 mM L-alanyl-L-glutamine...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...at RT. During the incubation, add 10 mL of DMEM Complete to a 15 mL conical and an appropriate amount of...the media. Replace the media with 10 mL of DMEM Complete or OptiPro SFM. Incubate the cells. Virus can ...

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

...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...