...bases 40-60% G/C content Start and end with 1-2 G/C pairs Melting temperature (Tm) of 50-60°C Primer pairs...in size. The structure of the primer should be relatively simple and contain no internal secondary structure...pairs should have a Tm within 5°C of each other Primer pairs should not have complementary regions Note:...

...thermocycler programmed to start at 95°C for 2 minutes. Then, gradually cool to 25°C over 45 minutes. Ligation Dilute... Let's assume that your favorite vector has a relatively limited MCS (BamHI - EcoRI - SalI) and you want...off the 3’ G on each oligo (and the complementary C of the other oligo), but this would destroy the EcoRI...oligos in a 1.5mL microfuge tube. Place tube in 90-95°C hot block and leave for 3-5 minutes. Remove the hot...

...Shaking incubator at 37 °C Stationary incubator at 37 °C Water bath at 42 °C Ice bucket filled with ice... storage at 4°C and let warm up to room temperature and then (optional) incubate in 37°C incubator. Mix...also make their own competent cells. This is a relatively simple procedure and is useful for performing...transform Procedure Take competent cells out of -80°C and thaw on ice (approximately 20-30 mins). Remove...placing the bottom 1/2 to 2/3 of the tube into a 42°C water bath for 30-60 secs (45 secs is usually ideal...without antibiotic) to the bacteria and grow in 37°C shaking incubator for 45 min. Pro-Tip This outgrowth...and won't grow in colonies. Incubate plates at 37°C overnight. Tips and FAQ How can I save time when carrying...

...parameters: Cycling Step Temperature (°C) Time (min) Ramp Rate (°C/sec) # Cycles Enzyme Activation 95 10...titer: $$T = {V*C*D\over v}$$ Where: T = Infectious titer, TU/mL V = Viruses per genome C = # Cells per ... across the dilutions. RPP30 copies should be relatively constant across samples. In the RRE example below...concentration of RPP30 positive droplets stays relatively even across samples (green). To increase the ...Bio-Rad, 1863051 8-strip PCR tubes, Axygen, PCR-02-FCP-C ddPCR 96-well PCR plates, Bio-Rad, 12001925 Pierceable...Benzonase in DMEM complete to each well. Incubate at 37 °C for 30 min. Gently aspirate media from wells, using...can be used for ddPCR immediately or stored at -20 °C until ready to use. Preparing for ddPCR Thaw samples...

... your experiment. Water baths tend to heat up relatively slowly. Place the water bath cover on the top... refrigerators to achieve a temperature between 4°C and room temperature. This general protocol suits ...

...be 10 µg/mL. Pro-Tip Transducing too many cells relative to the number of virus particles reduces the transduction...heat-inactivated FBS and 5mL of 100X glutaGRO. Store at 4 °C. Pro-Tip Different brands and FBS lots can promote...it can be inactivated in the lab by heating to 56 °C for 30 min. Considerations Before You Start The health...dose of antibiotic, which may not be stable at 37 °C. To achieve a stable cell pool, the antibiotic selection...well plate and left undisturbed for 13 days. (a, b, c) Colonies formed by expansion of single cells for ...antibiotic in culture, or remove the antibiotic entirely. If the antibiotic is reduced or removed from ...

...) or a similar photo software to determine the relative intensity of the protein bands to the overall ...standard Before Starting Warm the hot plate to 100 °C. Thaw IgG standard and prestained protein ladder on...microcentrifuge. Heat the samples for 10 min at 100 °C in a heat block. Spin the sample briefly in the microcentrifuge...

...hemocytometer or other cell counter. Pro-Tip Prepare a relatively dilute cell solution (<10 6 cells/mL) before ...heat-inactivated FBS and 5 mL of 100X glutaGRO. Store at 4 °C. Polyclonal stable cell pool: see our protocol for...

...with pLKO.1 C. Cloning shRNA oligos into pLKO.1 C.1 Recommended materials C.2 Annealing oligos C.3 Digesting...Virus may be stored at 4°C for a few days, but should be frozen at -20°C or -80°C for long-term storage...., R is a purine (A,G), and Y is a pyrimidine (C,U). G-C content should be 36-52%. Sense 3’ end should ...annealed oligo from step C.2 20 ng digested pLKO.1 TRC-cloning vector from step C.3 (If you were unable ...Store at 4°C. m. Add 5 mL of fresh media containing antibiotics to the cells and incubate at 37°C, 5% CO ...at 37°C, 5% CO 2 overnight. Day 2: b. The target cells should be approximately 80-90% confluent. c. Dilute...solution is stable at 4°C for up to one year. The powder form of polybrene is stable at 4°C for several years...

...instrument using SYBR detection: 98 °C 3 min / 98 °C 15 sec / 58 °C 30 sec / read plate/ repeat 39x from...and store at -20 °C. Once a standard is thawed do not freeze it again but store at 4 °C and use within 1...dilution. References Aurnhammer C, Haase M, Muether N, Hausl M, Rauschhuber C, Huber I, Nitschko H, Busch ...mix sample (do not vortex) Incubate 30 min at 37 °C Transfer to ice ** Critical: do NOT treat your plasmid...10 7 1.00 x 10 6 1.00 x 10 5 1.00 x 10 4 empty NTC C AAV reference Sample 3 D E Sample 1 Sample 4 F E Sample...presence of a second peak at a temperature of ~70–75 °C usually indicates the presence of primer dimers which...reference (typically ROX dye), to normalize non-PCR–related fluorescence fluctuations and to minimize well-...

...an incubation of 20 min to overnight at -20 °C or -80 °C will improve precipitation. Pour out the supernatant...Optional: Place the tube either at -20 °C overnight OR -80 °C for 30 min OR on dry ice for 5 min. Note...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 ...the supernatant in the new tube and incubate at 37 °C for 5 min. Note: Ribonuclease A (RNase A) is a pancreatic...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 the...

...media at 37 °C (for at least 15 minutes). Prewarm 3X LB Agar + Antibiotic plates at 37 °C. Prewarm 8X ...Chill a box of 200 µL micropipette tips in a -20 °C freezer. Aliquot 3 mL SOC into each of four 14 mL ...purification. Immediately purify them! Commercial Maxipreps rely on incremental, ordered cell lysis. E. coli cells...digest. Recombination is thought to be in part related to selective pressure. These recombined plasmids...can dramatically plateau and sometimes fall off entirely if the column or reagents are significantly overloaded...