...pH 8) 50 mM glucose 10 mM EDTA Store Solution I at 4°C Solution II - Denaturing Solution 0.2 N NaOH 1.0%...acetic acid 57 mL of dH 2 O Store Solution III at 4°C Grow 2 mL overnight cultures from single colonies...to the recovered aqueous DNA layer. Repeat steps 2-4. Note: Phenol-chloroform is a hazardous waste - DO...high speed (at least 12,000 rpm) for 15-30 min at 4°C. Notes: Pellet contains the precipitated DNA. Supernatant...temperature before quantifying and using. Store DNA at 4°C. Tips and FAQ Plasmid purification kits provide ... supernatant out of the tube if you are worried about losing the pellet. Dry with vacuum or by inverting...

...Reagent Preparation DMEM Complete: 10% v/v FBS and 4 mM L-alanyl-L-glutamine (or stable alternative, such...heat-inactivated FBS and 5 mL of 100X glutaGRO. Store at 4 °C. Polyclonal stable cell pool: see our protocol ...solution. Homogenized cell solution concentration: 4 × 10 5 cells/mL To seed one 96-well plate, make 10... of homogenized cell solution needed: (50 cells)/(4 × 10 5 cells/mL) = 0.125 µL Because this is such a...Shalem O, Zhang F. Nature Methods. 2014 Aug;11(8):783-4. (Link opens in a new window) Last reviewed on: September...approximately 50–60% confluent. For 293T cells this is about 2 × 10 6 cells in a 10 cm dish. Each 10 cm dish ...cell solution into the conditioned medium prepared above to make a new cell solution at a concentration of...

...and transduce cells Day 4: Treat cells with Benzonase and harvest cells Day 4+: ddPCR and analysis Equipment...VIC) 1 µL 9 µL 900 nM, 250 nM Nuclease-free water 4 µL 36 µL Total Volume 16 µL 144 µL Vortex the master... collecting drops along the side of the tube. Add 4 µL of the 25 ng/µL samples to the appropriate PCR ...Extension 60 1 2 40 Enzyme Deactivation 98 10 2 1 Hold 4 ∞ 2 1 After PCR is complete, transfer the plate to...0.08366013072 6.69E+06 3 200 1620 15840 0.2045454545 8.18E+06 4 100 3180 20540 0.3096397274 6.19E+06 5 50 8960 17080...the titer. Calculations In the experimental setup above, the following virus dilutions were used to transduce...v = Virus volume, mL *In the experimental setup above, the cells per well is 300,000 and the virus volume...

...Moffat laboratory members conducted multiple screens in different human cell models and identified ~4-5-fold... the MAGE method in unmodified bacterial species (4). This set of plasmids (dubbed pORTMAGE) expresses...but be sure to let us know if you'd like to write about your plasmids in a future blog post. No time to ...pooled libraries, but thanks to a deposit from the laboratory of Dr. Ji-Long Liu at Oxford University, we are...you plan to use GMAP for your experiments, think about depositing your new plasmids with Addgene to help...and context-dependent human fitness genes, the laboratory of Jason Moffat at the University of Toronto ...This work has been published in Cell. The Moffat laboratory maintains a website for the TKO library that ...

...2: Harvest cells and purify DNA (Estimated time 3-4 hours) Cells should be harvested first thing in the... Reagents 200 µL electrocompetent cells (Default: 4 tubes of Endura Duos, Lucigen, 60242-1) Alternatives...Agar + Antibiotic 65 mm Petri dish (VWR, 11019-552) 4 MaxiPreps (Qiagen HiSpeed Max, Catalog #12663) Tips...plates. Prechill Micropulser cuvettes on ice. Thaw 4 tubes of electrocompetent cells on ice for 15-20 minutes... incubation if needed. Place 100 mL sterile LB at 4 ℃. Day 2 (morning) Before beginning, prechill at least... LB-bacteria per plate. Centrifuge tubes (4000 G, 4 ℃, 15 minutes) to pellet bacteria. Decant LB and weigh...require modifications dictated by the originating laboratory for optimal results. If you obtained the pooled...

...Prepare carbenicillin to a concentration of 1 mg/mL – 4 mg/mL in LB medium. The concentration of antibiotic... 1 mg/mL plate and effective selection. 150 µL of 4 mg/mL Carbenicillin plated over-agar Plate shows several...coli after Over-Agar Plating of Carbenicillin. The above graph displays the stock concentration of Carbenicillin...concentrations that will work for this assay, and the above result represents a single experiment. For publishable...

...through the expression of transcription factors OCT3/4, SOX2, KLF4, and c-MYC is the established model for...but be sure to let us know if you'd like to write about your plasmids in a future blog post. No time to ...repository. These variants can be used as tools to learn about the influence of structure on an FP’s properties...100bp or 1kb fragments are produced (see figure above). 3 Reasons The PSU Ladders Are Awesome Tools: ...

...Reagent Preparation DMEM Complete: 10% v/v FBS and 4 mM L-alanyl-L-glutamine (or stable alternative, such...heat-inactivated FBS and 5mL of 100X glutaGRO. Store at 4 °C. Pro-Tip Different brands and FBS lots can promote...Shalem O, Zhang F. Nature Methods. 2014 Aug;11(8):783-4 (Link opens in a new window) . Last reviewed on: September...cells in the untransduced well (0 µL lentivirus, above) are dying. Perform regular media changes and monitor...

...out the following into a 500 mL glass bottle: 4 g NaCl 4 g Tryptone 2 g Yeast Extract and dH 2 O to 400...suggested amount, instead of the other dry ingredients above. Media without growth (top) and with growth (bottom...

...gel with a pipette tip. Place newly poured gel at 4 °C for 10-15 mins OR let sit at room temperature for...will set more quickly if you place the gel tray at 4 °C earlier so that it is already cold when the gel...cool down to about 50 °C (about when you can comfortably keep your hand on the flask), about 5 mins. Optional...concentration of approximately 0.2-0.5 μg/mL (usually about 2-3 μl of lab stock solution per 100 mL gel). EtBr... of the tip of the pipette into the buffer just above the well. Very slowly and steadily, push the sample... of the way down the gel. A typical run time is about 1-1.5 hours, depending on the gel concentration ...

...): 5 µL in 95 µL 1X PCR buffer (1:8,000) Dilution 4 (20X): 5 µL in 95 µL 1X PCR buffer (1:160,000) Dilution...Extension 60 1 2 50 Signal Stabilization 98 10 2 1 Hold 4 ∞ 2 1 After PCR is complete, transfer the plate to...factor of virus V = Volume of virus in reaction mix (4 µL) Sample Data When analyzing data there should be...dilution BSC. Prepare 1X dilution buffer (see recipe above). Save ~25 µL of 1X dilution buffer in a microcentrifuge...

...used to denote that the position could be any of the 4 bases. Check the trace file and see if you can manually...Addgene lists the primers used to obtain each result above the posted sequence in the "View Sequence" link....sequence than expected and wish to contact Addgene about the accuracy of your plasmid, please email help@...

...walk-in refrigerators to achieve a temperature between 4°C and room temperature. This general protocol suits... lab! Introduction A water bath is a piece of laboratory equipment that helps bring your materials to ...even if you are using disinfectants as described above in step 3. You will also need to maintain the appropriate...

...vector in molar ratios (vector:insert) between 4:3 and 1:6 in a standard ligation reaction (ex. to ligate ...water and quantify the concentration (should be about 8ng/μl). Mix the annealed oligos with cut vector...

...cloning, it is recommended that you digest for at least 4 hours. If you will be using the digested DNA for another...more at (Link opens in a new window) NEB's website about star activity . If you are digesting a large number...

...therefore it is important that the digest go at least 4 hours and as long as overnight. If you are going to...Information about plasmid cloning by restriction enzyme digest (subcloning), including design and experimental...window) New England Biolabs for more information about restriction enzyme buffers). If you select enzymes...

...DNA strand accurately and rapidly. Repeat steps 2-4 25-30 times. Final Extension for 5 minutes at 72°C...manufacturer’s instructions for specific instructions about extension time and temperatures. Initial Denaturation...

...therefore it is important that the digest goes at least 4 hours and as long as overnight. If you are going to...Background In its simplest form, PCR based cloning is about making a copy of a piece of DNA and at the same ...