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We narrowed to 29 results for: POR C

Showing: 1 - 20 of 29 results

  1. Protocol - pLKO.1 – TRC Cloning Vector

    Type
    Protocol
    ...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...

  2. Kit Free RNA Extraction

    Type
    Protocol
    ...before you start. Homogenizer Vortexer -20°C freezer -80°C freezer Reagents Solution D (for Protocol Option...instead of incubating at -20°C for 1 hour, you can try incubating at -80°C overnight. Centrifuge for 20...centrifuge the sample(s) for 15 minutes at 12,000 × g at 4°C to separate RNA from the rest of the tissue/cell lysate... lysate. Pro-Tip Having your samples spin at 4°C helps reduce RNA degradation. If you don’t have access...Isopropanol to the extracted aqueous layer. Incubate at -20°C for 1 hour. Lithium Chloride (Option B): LiCl selectively...extracted aqueous layer to 2.5 M. Incubate at -20°C for 1 hour. Pro-Tips If you anticipate your RNA yield...

  3. What is Polymerase Chain Reaction (PCR)

    Type
    Protocol
    ...minutes at 94°C. Denature for 30 seconds at 94°C. Anneal primers for 30 seconds at 55°C (or 5°C below Tm)....step is commonly performed at 94–98°C. Denature 30 seconds at 94°C: Continued denaturation of double stranded...for 1 minute at 72°C: The Taq polymerase has an optimal temperature around 70-75°C so this step enables...minutes at 72°C. Repeat steps 8-10 for 25-30 cycles. Final Extension for 5 minutes at 72°C. Run 2 μL on...requires free nucleotides [dNTPs; adenine (A), cytosine (C), guanine (G), thymine (T)] in an equal molar ratio... Program Initial Denaturation for 2 minutes at 94°C: This initiation step heats the double stranded DNA...stranded DNA. Anneal primers for 30 seconds at 55°C: The forward and reverse primers are stable within...

  4. Protocol - How to Create a Bacterial Glycerol Stock

    Type
    Protocol
    ... can open unexpectedly at -80°C. Freeze the glycerol stock tube at -80°C. The stock is now stable for ...sample at -80°C. Frozen tubes are hard to write on and samples stored for long periods at -80°C can lose labels...are important for long-term storage of plasmids. Although you can store your plasmid DNA at -20°C, many.... Bacteria on an LB agar plate can be stored at 4°C for a few weeks. However, if you want to store bacteria...glycerol stock of bacteria can be stored stably at -80°C for many years. Protocol Video Watch the protocol ...for years, as long as it is kept at -80°C. Subsequent freeze and thaw cycles reduce shelf life. To recover...completely and will improve the shelf life. It is very important that you shake the glycerol before freezing (5...

  5. Transfection for Recombinant Antibodies

    Type
    Protocol
    ...Automated cell counter 37 °C, 5% CO 2 incubator with shaking platform set to 120 rpm 37 °C bead bath Vortex Stir...: Harvest antibody Equipment Biosafety Cabinet 4 °C Refrigerator Micropipettes and tips Pipettor and pipettes...BalanCD HEK293 transfection media (BCD TFX) to 37 °C. See below for other Reagent Preparation instructions...Prepare 10 mL aliquots and store the solution at -20 °C. 1 mg/mL PEI-MAX Add 1 g of PEI-MAX powder to 900 ....22 µm filter. Prepare aliquots and store at -20 °C until use. BCD TFX 1000 mL BalanCD HEK293 Media 10...Glutagro Do not add selective reagents Store at 4 °C until use. We suggest preparing fresh solutions after...BalanCD HEK293 Feed 20 mL 200 mM Glutagro Store at 4 °C until use. We suggest preparing fresh solutions after...

  6. AAV Purification by Iodixanol Gradient Ultracentrifugation

    Type
    Protocol
    ...min at 4 °C and discard the flow through. Add your sample. Spin at 3500 rpm for 5–8 min at 4 °C, discard...possible. Store at 4 °C for short term (2 weeks), or aliquot and store at -80 °C for long term. Sample... passing through a 0.22 μm filter and store at 4 °C. 1X PBS-MK buffer Dissolve 26.3 mg of MgCl 2 , and... passing through a 0.22 μm filter and store at 4 °C. 0.1% Pluronic-F68 in PBS (A) Add 500 µL of 10% Pluronic-F68... mL PBS 0.001% Pluronic-F68 in PBS + 200 mM NaCl (C) (formulation buffer) Add 5 mL of Buffer B and 2 mL... at 350,000 x g for 90 min in a T70i rotor at 10 °C. Pro-Tip If you need more time, you can alternatively...alternatively centrifuge for 2 h at 200,000 x g at 18 °C. Carefully take the QuickSeal tubes out of the rotor ...

  7. Protocol - Bacterial Transformation

    Type
    Protocol
    ...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...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.... Transformation of bacteria with plasmids is important not only for studies in bacteria but also because...

  8. AAV Titration by qPCR Using SYBR Green Technology

    Type
    Protocol
    ...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...vector cores. Our titers were similar to those reported by these institutions. This protocol is for a ...

  9. AAV Production in HEK293 Cells

    Type
    Protocol
    ... Store the tubes at -80 °C. After thawing, the solution can be stored at 4 °C for up to 2 months. After.... Add stir bar and stir slowly at 4 °C for 1 h, then keep at 4 °C for 3 h without stirring to allow full...it can be inactivated in the lab by heating to 56 °C for 30 minutes. 0.45 μm polyethersulfone (PES) filter...11 mL of 200 mM L-alanyl-L-glutamine. Store at 4 °C. D1 + 0.1 M sorbitol (optional) : DMEM low glucose...may separate into phases. Aliquot and store at 4 °C. Cell Lysis Buffer : 50 mM Tris HCl, 150 mM NaCl,...sterilize through a 0.22 μm membrane. Store at 4 °C. Considerations Before You Start The health of the...and cells. Centrifuge at 3900 rpm for 20 min at 4 °C to pellet the cells. Keep the cell pellet on ice and...

  10. Lentivirus Production

    Type
    Protocol
    ...Store the tubes at -20 °C or -80 °C. After thawing, the solution can be stored at 4 °C for up to 2 months....heat-inactivated FBS and 5 mL of glutaGRO. Store at 4 °C. Pro-Tip Different brands and lots of FBS can promote...it can be inactivated in the lab by heating to 56°C for 30 min. 1 mg/mL polyethylenimine, linear MW 25,000... tissue culture plates. Incubate the cells at 37 °C, 5% CO 2 for ~20 h. Prepare a mixture of the three...media to a polypropylene storage tube and store at 4 °C between harvest. Centrifuge the viral supernatant ...filter. The viral supernatant can be stored at 4 °C for several hours but should be aliquoted and snap...snap frozen in liquid nitrogen and stored at -80 °C as soon as possible to avoid loss of titer. Sample Data...

  11. Protocol - How to Design Primers

    Type
    Protocol
    ...bases 40-60% G/C content Start and end with 1-2 G/C pairs Melting temperature (Tm) of 50-60°C Primer pairs...pairs should have a Tm within 5°C of each other Primer pairs should not have complementary regions Note:...toward one another. The size of the primer is very important as well. Short primers are mainly used for amplifying...

  12. Protocol - How to Streak a Plate

    Type
    Protocol
    ...hours) at 37 °C. Pro-Tip Some plasmids or bacteria need to be grown at 30 °C instead of 37 °C. This is often...unstable plasmids, which sometimes recombine at 37 °C. Be sure to check this before incubating your plate...initials. Labeling within a laboratory setting is important for organization, and it is recommended that you...

  13. Antibody Validation Using the Indirect ELISA Method

    Type
    Protocol
    ... incubating certain steps at 4 °C instead of room temperature or 37 °C. The protocol notes when there ...well plate seal and incubate at 37 °C for 30 min , or overnight at 4 °C . Section 2: Block the plate Prepare...rpm for 2 h at room temperature or overnight at 4 °C . Section 3: Primary antibody incubation Carefully...rpm for 2 h at room temperature or overnight at 4 °C . Section 4: Secondary antibody incubation Carefully...rpm for 2 h at room temperature or overnight at 4 °C . Section 5: TMB reaction Carefully remove the plate...that sharing the full details of our protocols supports reproducibility and accelerates science. Here,...information is solely for transparency intended to support reproducibility in science. General Considerations...

  14. Western Blot

    Type
    Protocol
    ...Spatula Platform shaker Cold room Gel imager -80 °C freezer Reagents 1X PBS Lysis buffer e.g., RIPA lysis... Centrifuge lysate for 15 min at 14,000 x g at 4 °C . Transfer supernatant to a clean microcentrifuge ...tube. Use the lysate immediately or store at -80 °C until ready to use. Section 2: Determine the total...well microtiter plate. Incubate for 30 min at 37 °C . Determine the absorbance at 590 nm . Calculate the...sample to 1X . Boil the samples for 10 min at 100 °C . Section 3: SDS-PAGE Prepare the precast gel as follows... the membrane overnight in primary antibody at 4 °C on a rocking platform. Pro-Tip Primary antibody incubation...that sharing the full details of our protocols supports reproducibility and accelerates science. Here,...

  15. Affinity Purification of Recombinant Antibodies with Protein A or Protein G

    Type
    Protocol
    ...spectrophotometer 37 °C, 5% CO 2 incubator with shaking platform set to 120 rpm 37 °C bead bath Clamp stand...Equipment Class II, Type A2 Biological Safety Cabinet 4 °C Refrigerator Pipette controller Benchtop centrifuge...phosphate dibasic (NaH 2 PO 4 ) Store up to 1 month at 4 °C Adjust pH to 7.0 Autoclave or filter sterilize Protein...µm PES filter. Aliquot and freeze upright at -20 °C Procedure Section 1: Affinity chromatography Equilibrate...A/G binding buffer and store in 20% ethanol at 4 °C . Pro-Tip Columns may be re-used up to 5x when purifying...446 Benzamide, Millipore Sigma 12072 Antipain, Millipore Sigma 10791 Leupeptin, Millipore Sigma L2884 Aprotinin...monobasic monohydrate, Millipore Sigma 71507-250G Sodium phosphate dibasic, Millipore Sigma S7907-500G 1 ...

  16. Protocol - How to Inoculate a Bacterial Culture

    Type
    Protocol
    ...at 37°C for 12-18 hr in a shaking incubator. Note: Some plasmids or strains require growth at 30°C. If ...more slowly. Also, bacteria incubated at 30°C rather than 37°C often require longer incubation times. Double...plasmid. However, a liquid culture is capable of supporting a higher density of bacteria and is used to grow...

  17. Lentivirus ddPCR Titration

    Type
    Protocol
    ...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 ...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...EX0276-1 Benzonase 250 U/µl, Millipore #71205-3 Polybrene 10 mg/mL, Millipore, TR-1003-G Molecular Biology...place the PCR plate with the foil onto the metal support block. Place the block in the plate sealer and ...

  18. Protocol - How to Run an Agarose Gel

    Type
    Protocol
    ...attention. Let agarose solution cool down to about 50 °C (about when you can comfortably keep your hand on ... with a pipette tip. Place newly poured gel at 4 °C for 10-15 mins OR let sit at room temperature for ... set more quickly if you place the gel tray at 4 °C earlier so that it is already cold when the gel is...period of time; b) using a wider/thinner gel comb; or c) loading less DNA into the well. Another method for... be in the bottom portion of the gel, but all of the EtBr will be in the top portion and your bands will...overboil the solution, as some of the buffer will evaporate and thus alter the final percentage of agarose...

  19. Protocol - How to Ligate Plasmid DNA

    Type
    Protocol
    ...individual tubes of 5, 10 or 20μL for storage at -20°C. Whenever you need to set up ligations in the future...optimization. Incubate at room temperature for 2hr, or at 16°C overnight (following the manufacturer’s instructions...efficiency of ligation can be improved by incubation at 37°C. Proceed with bacterial transformation . Tips and ...desired insert. If you are in this situation, it is important to treat the digested vector backbone with a phosphatase...setting up the ligation reaction itself, it is important to determine the amount of cut insert and vector...being sure to increase the amount of buffer proportionally. 1μL of ligase should be sufficient for larger...

  20. Virus Protocol - Generating Stable Cell Lines

    Type
    Protocol
    ...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 ...CI) Heat-inactivated FBS Polybrene (10 mg/mL), Millipore TR-1003-G 1X PBS pH 7.4 without calcium or magnesium...of cell death upon antibiotic selection. It is important to monitor these cells regularly and replace the... the surviving cells in the culture, so it is important to do regular media changes and maintain optimal...
Showing: 1 - 20 of 29 results