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We narrowed to 395 results for: POR C

Showing: 361 - 390 of 395 results

  1. 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...

  2. 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...

  3. 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...

  4. 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 ...

  5. 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...

  6. Adenovirus Guide

    Type
    Guide
    ...new window) Lee, C. S., Bishop, E. S., Zhang, R., Yu, X., Farina, E. M., Yan, S., Zhao, C., Zheng, Z., Shu...Yang, C., Wu, K., Wu, Y., Ho, S., Athiviraham, A., Lee, M. J., Wolf, J. M., Reid, R. R., & He, T. C. (2017... A., Luu, H. H., Haydon, R. C., Kinzler, K. W., Vogelstein, B., & He, T. C. (2007). A protocol for rapid... B. L., Corder, B. N., Gordon, D. N., Pierson, T. C., & Weaver, E. A. (2020). Characterization of a Species...K. J., Sebastian, S., Spencer, A. J., Gilbert, S. C., Hill, A. V. S., & Lambe, T. (2017). Chimpanzee adenoviral...Geisbert, T. W., Bailey, M., Hensley, L., Asiedu, C., Geisbert, J., Stanley, D., Honko, A., Johnson, J...21325402 (Link opens in a new window) Gilbert, S. C. (2015). Adenovirus-vectored Ebola vaccines . Expert...

  7. 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 ...

  8. 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...

  9. 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...

  10. 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...

  11. Adeno-associated virus (AAV) Guide

    Type
    Guide
    ...Michael, S., Kuleshova, E., Ojala, D., Huang, C. C., Gerfen, C. R., Schiller, J., Dudman, J. T., Hantman,...Technology References Burger, C., Gorbatyuk, O. S., Velardo, M. J., Peden, C. S., Williams, P., Zolotukhin... Ravi, N., Wu, W. L., Sánchez-Guardado, L., Lois, C., Mazmanian, S. K., Deverman, B. E., & Gradinaru, ...) Liu, D., Li, T., Liu, L., Che, X., Li, X., Liu, C., & Wu, G. (2024). Adeno-associated virus therapies... to display neuronal retrograde transport, AAVrg. Retrograde transport refers to the direction of movement...nuclear export. Poly(A) signal in cis The polyadenylation or poly(A) signal aids in the nuclear export of ...soma to axon) and retrograde (from axon to soma) transport. Many rAAV vectors have been engineered to express...

  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. Lentiviral Vector Guide

    Type
    Guide
    .... M., Chen, I. S., Hahn, W. C., Sharp, P. A., Weinberg, R. A., & Novina, C. D. (2003). Lentivirus-delivered...PMID: 33768124 Marshall, H. M., Ronen, K., Berry, C., Llano, M., Sutherland, H., Saenz, D., Bickmore, ...PMID: 18092005 Merten, O., Hebben, M., & Bovolenta, C. (2016). Production of lentiviral vectors. Molecular...Schambach, A., Zychlinski, D., Ehrnstroem, B., & Baum, C. (2013). Biosafety features of lentiviral vectors....., Kochenderfer, J. N., Norberg, S. M., Hinrichs, C., Highfill, S. L., Somerville, R. P., Panch, S. R....transcription of the full-length virus after it is incorporated into a host genome. This is often found in second-generation...cells, they kill off any cells that have not incorporated the lentiviral genome and the cells that survive...

  14. 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...

  15. 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,...

  16. 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 ...

  17. 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...

  18. Gamma-Retroviral Vector Guide

    Type
    Guide
    ...Wong, G. K., Cameron, E. R., Kilbey, A., & Neil, J. C. (2016). Gamma-Retrovirus integration marks cell Type-Specific...10.1371/journal.pone.0154070 PMID: 27097319 LaFave, M. C., Varshney, G. K., Gildea, D. E., Wolfsberg, T. G....gkt1399 PMID: 24464997 Maetzig, T., Galla, M., Baum, C., & Schambach, A. (2011). Gammaretroviral vectors:...Rubat, L., Nikoniuk, A., Macmorland, W., Horlock, C., Matsumoto, S., Williams, S., Smith, K., Price, J...cells, they kill off any cells that have not incorporated the retroviral genome and the cells that survive...the only type of selectable marker. Fluorescent reporters, such as GFP, are another common selection marker...interest and contains the sequences that will be incorporated into the host cell genome but cannot produce...

  19. 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 ...

  20. Chemogenetics Guide

    Type
    Guide
    ...neuronal firing through stimulating phospholipase C, which releases intracellular calcium stores. There...Mimura K, English JG, Liu J, Inoue K, Kumata K, Seki C, Ono M, Shimojo M, Zhang M-R, Tomita Y, Nakahara J...Link opens in a new window) Vardy E, Robinson JE, Li C, Olsen RHJ, DiBerto JF, et al. (2015). A New DREADD...Haberman A, Graham J, Block J, Zhou W, Chen Y, Zhang S-C (2021). Human Stem Cell-Derived Neurons Repair Circuits...control expression of a chemogenetic reporter ensures that the reporter remains silent until a cell or tissue-specific...ligands can have high potency, which is potentially important for dosing in animal studies, made them attractive...agonists for PSAM4. A given PSAM is coupled with an ion pore domain (IPD) to form ligand-gated ion channels (...

  21. 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...

  22. 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...

  23. 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...

  24. Gibson Assembly Protocol

    Type
    Protocol
    ... Cepko, 2020). Incubate the mix for 1 hour at 50 °C or follow manufacturer's instructions. You can purchase...Gibson DG, Smith HO, Hutchison CA, Venter JC, Merryman C. (2010). Chemical synthesis of the mouse mitochondrial....1515 (Link opens in a new window) Rabe BA, Cepko C. (2020). A Simple Enhancement for Gibson Isothermal...other. Phusion High-Fidelity DNA Polymerase - incorporates nucleotides to “fill in” the gaps in the annealed... product by restriction digest . Sequence the important regions of your final plasmid, particularly the...

  25. AAV ddPCR Titration

    Type
    Protocol
    ...parameters. Cycling Step Temperature (°C) Time (min) Ramp Rate (°C/sec) # Cycles Denaturation 95 10 2 1 ...sample: $$T = {R*C*D*1000\over V}$$ Where: T = Titer, GC/mL R = Reaction volume (20 µL) C = Copies/µL D ...Bio-Rad, 1863051 8-strip PCR tubes, Axygen, PCR-02-FCP-C ddPCR 96-well PCR plates, Bio-Rad, 12001925 48-well...place the PCR plate with the foil onto the metal support block. Place the block in the plate sealer and ... reading. When the droplet reading is complete, export the data from all wells as a CSV file and use the...

  26. Antibody Guide

    Type
    Guide
    ...stored at 4 °C for short-term storage. For long-term storage they should be stored at -20 °C in aliquots...conjugated to fluorophores should never be stored below 4 °C. Certain isotype subclasses, such as IgG3, aggregate... after thawing and therefore should be kept at 4 °C. Record and track lot numbers for all antibodies used...necessary in cases where extreme specificity is important. Their epitope specificity allows binding to a...antibodies only bind a single epitope, so it is important to choose a monoclonal antibody that will recognize...using a secondary against a tag, such as His, incorporated in the sdAb. sdAbs can have weak signals due...with conjugating reactions are possible. It is important to research your reaction and buffer before conjugating...

  27. Optogenetics Guide

    Type
    Guide
    ... PMID 22341318 Gradinaru V, Zhang F, Ramakrishnan C, Mattis J, Prakash R, Diester I, Goshen I, Thompson...20621963 Mattis J, Tye KM, Ferenczi EA, Ramakrishnan C, O'Shea DJ, Prakash R, Gunaydin LA, Hyun M, Fenno ...neural populations at distinct times. Temporal considerations. Temporal precision is key in optogenetic experiments... exposed to light of the correct wavelength, the pore opens, cations flow into the cell (yellow dots),...optical switches Sensors are genetically-encoded reporters of molecular signals; e.g., calcium indicators...specific subsets of neurons, allowing precise spatiotemporal control of these neurons by turning on and ...addition of trafficking signal from Kir2.1 and ER export signal provide improved membrane targeting 589 ...

  28. Fluorescence Titering Assay

    Type
    Protocol
    ...heat-inactivated FBS and 5 mL of 100X 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. Considerations Before You Start The health...CI) Heat-inactivated FBS Polybrene (10mg/mL), Millipore TR-1003-G 1X PBS pH 7.4 without calcium or magnesium...

  29. CRISPR Library Amplification

    Type
    Protocol
    ...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 ... mix. Electroporate cells (one at a time for a total of eight electroporations): Electroporator Conditions...and have 1 mL SOC per electroporation readily available for post-electroporation recovery of cells. Ensure...if the recombinant band makes up a significant proportion of the DNA pool. Last Update: August 17, 2023...limited. Equipment Table top centrifuge BioRad Electroporator (MicroPulser TM , Bio-Rad 1652100) Reagents.../or increasing the skewness of the pool. 8 electroporation cuvettes (BioRad, Micropulser, 0.1 cm ) 20 ...

  30. Colony Formation Titering Assay

    Type
    Protocol
    ...inactivated FBS and 5 mL of 100X 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. Considerations Before You Start The health...CI) Heat-inactivated FBS Polybrene (10 mg/mL), Millipore TR-1003-G 1X PBS pH 7.4 without calcium or magnesium...
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