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We narrowed to 698 results for: des.1

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  1. Using Video to Share Your Science: We Share Science

    Type
    Blog Post
    ...email Ryan at [email protected]   References 1. Larivière, Vincent, Yves Gingras, and Éric Archambault.... After months, or years, of grueling effort to design, fund, and conduct a research project, the vast...provide more than enough time for researchers to describe their science, and for viewers to decide if reading...is of interest. In his research, Scott Spicer concludes that “… the emergence of the video abstract genre...websites). WeShareScience is a social platform that provides links to the videos, keeping the copyright and...

  2. Binning Singletons: Tackling Conference Networking When You Don’t Know Anyone

    Type
    Blog Post
    ...conference for any reason was a Singleton. Figure 1: The Binning Singletons logo. Image: James et al.,...was that kid who used recycled notebook paper, despite the fact that it was gray. He has worked in molecular...molecular and microbiology for the last two decades as a staff scientist, but before going to graduate ...

  3. Plasmids 101: Stringent Regulation of Replication

    Type
    Blog Post
    ...levels of DnaA to get the job done.   Figure 1: DNA melting by DnaA and IHF is the first step of ...replicate—but not always to the same extent. As described in our previous Origin of Replication post, DNA...replication. For example, the plasmid ColIb-P9 encodes the protein RepZ to drive its own replication. ...

  4. Antibodies 101: Beyond Surface Labeling

    Type
    Blog Post
    ... panel design. Find streptavidin plasmids and biotin plasmids at Addgene!     Figure 1: Indirect... this system is increased flexibility for panel design. Another perk is that one primary antibody can ...for example, a previously induced gene knockout. Besides amine- and DNA-binding dyes, nucleoside analogs...walk through the topics we covered in this post. Besides direct labeling, indirect labeling can be used ...surface, dump gating is a useful way to get rid of undesired cell populations. Dump gating uses the same color...

  6. Transferable Skills Guide: External Collaborations

    Type
    Blog Post
    ...collaborations, keep these jobs in mind! References 1. Sinche, Melanie, et al. "An evidence-based evaluation...collaborators than you realize. Being at a university provides the perfect platform for practicing initiative...

  7. Transferable Skills Guide: Conflict Resolution

    Type
    Blog Post
    ...’re not one of these people yourself.  Fig 1: Quote from Michael Ende, courtesy of morefamousquotes.com...even better. The Thomas Kilmann Conflict Model describes 5 basic approaches to conflict resolution based...

  8. Finding Your Perfect Job After University

    Type
    Blog Post
    ...experience in cancer research After graduating with a 2:1 BSc in Molecular Biology (roughly a B average in the...every item I tested was constantly tracked with barcodes and spreadsheets. It was great to be in the lab...industry positions. There were, however, some downsides to working in a large commercial company. In my...aspects of the company and gave me the freedom to design new projects and grow in the role. I understand... job different every day. I love the freedom to design and develop my own projects, but working remotely...

  9. A Guide to Getting Started in Undergrad Research

    Type
    Blog Post
    ...Bachelor's degree Generally a short-term position (1-2 years) Often a gap-year position taken to transition... are more involved in the science, experimental design, and execution. Can lead to an independent project...advice Give yourself permission to pursue your own desired path. Careers in science are also not limited to...

  10. Protocol - pLKO.1 – TRC Cloning Vector

    Type
    Protocol
    ...of Contents A. pLKO.1-TRC Cloning Vector A.1 The RNAi Consortium A.2 Map of pLKO.1 A.3 Related plasmids...Order oligos compatible with pLKO.1 C. Cloning shRNA oligos into pLKO.1 C.1 Recommended materials C.2 Annealing... References H.1 Published articles H.2 Web resources I. Appendix I.1 Sequence of pLKO.1 TRC-Cloning Vector...information Back to Top A. pLKO.1-TRC Cloning Vector A.1 The RNAi Consortium The pLKO.1 cloning vector is the backbone...marker encoded in pLKO.1 allows for convenient stable selection. Figure 1 : Map of pLKO.1 containing an shRNA...puromycin should be from 1-10 μg/mL in 1 μg/mL increments. d. Label plates from 1-10 and add appropriate...Appendix I.1. Sequence of pLKO.1 TRC-Cloning Vector Click here to see the sequence of pLKO.1 TRC-cloning...

  11. General Transfection

    Type
    Protocol
    ...Volume of 1 mg/mL PEI (μL) 1:1 18.9 18.9 1:2 18.9 37.8 1:3 18.9 56.7 1:4 18.9 75.6 1:5 18.9 94.5 1:6 18.9...were transfected using 1:1, 1:2, 1:3 and 1:6 µg of pRosetta :µg of PEI. The 1:2 and 1:3 ratios provided high.... Thawed aliquots should be discarded after 1–2 months. 1 mg/mL polyethylenimine, linear MW 25,000 Da ...stable. Dilute 1:3 (µg DNA:µg PEI) in 500 µL total of OptiPro SFM (per 10 cm plate). 56.7 µL of 1 mg/mL PEI...subclone of HEK293T optimized for viral production) Day 1: Transfect Cells Day 2 (am): 18 h post transfection...to be empirically determined for each new batch of 1 mg/mL PEI prepared. There may be variation between...plasmid using a variety of ratios. Check the cells 1–2 days after transfection to determine what ratio ...

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

    Type
    Protocol
    ...NaH 2 PO 4 ∙H 2 O 1 L deionized water Adjust pH to 7.0 Autoclave or filter sterilize 1 M sodium phosphate...NaH 2 PO 4 ∙H 2 O 1 L deionized water Adjust pH to 7.0 Autoclave or filter sterilize 1 M of sodium phosphate...protease inhibitor cocktail. Add 1 part Protein A/G binding buffer to 1 part tissue culture supernatant...Choose Option 1 or Option 2 based on the concentration of the pooled sample above. Option 1: Buffer exchange...antibody to 1 mg/mL with PBS if needed. For long term storage, add sterile sodium azide to 1 mM. Option... proceed to Option 1 with a buffer exchange using a Zeba Spin 7 kDa MWCO desalting column. If the concentration...purify recombinant antibodies. Workflow Timeline Day 1: Purify antibody Day 2 or later: Buffer exchange Equipment...

  13. Lentivirus Production

    Type
    Protocol
    ...DNA μL of 1 mg/mL PEI 1:1 18.9 18.9 1:2 18.9 37.8 1:3 18.9 56.7 1:4 18.9 75.6 1:5 18.9 94.5 1:6 18.9 113.4...μg total DNA to μg PEI ratios of 1:1, 1:2, 1:3 and 1:6. The 1:2 and 1:3 total DNA:PEI μg ratios provided...: Monday: Plate 1×10 6 cells in a T75 flask in 15 mL DMEM Complete. Wednesday: Plate 1×10 6 cells in a...Workflow Timeline Day 0: Seed 293T packaging cells Day 1 (pm): Transfect packaging cells Day 2 (am): 18 h post-transfection...inactivated in the lab by heating to 56°C for 30 min. 1 mg/mL polyethylenimine, linear MW 25,000 Da (PEI) ...to be empirically determined for each new batch of 1 mg/mL PEI and for each cell line. Considerations Before...enough PEI such that the ratio of μg DNA:μg PEI is 1:3 (1000 μL total per 10 cm dish). Using transfer plasmid...

  14. AAV ddPCR Titration

    Type
    Protocol
    ...Dilution 1 (20X): 5 µL in 95 µL 1X PCR buffer (1:20) Dilution 2 (20X): 5 µL in 95 µL 1X PCR buffer (1:400)...95 10 2 1 Denaturation 95 0.5 2 50 Annealing/Extension 60 1 2 50 Signal Stabilization 98 10 2 1 Hold 4 ...Use a single channel 1–10 µL pipette to add 5 µL of each viral sample to Dilution 1 in the 48-well dilution... 95 µL 1X PCR buffer (1:8,000) Dilution 4 (20X): 5 µL in 95 µL 1X PCR buffer (1:160,000) Dilution 5 (20X...95 µL 1X PCR buffer (1:3,200,000) Dilution 6 (2X): 50 µL in 50 µL 1X PCR buffer (1:6,400,000) Dilution ...50 µL 1X PCR buffer (1:12,800,000) Dilution 8 (2X): 50 µL in 50 µL 1X PCR buffer (1:25,600,000) Use multichannel...pipettes for the dilution series. For dilutions 1–5, use the 1–10 µL multichannel pipette set to 5 µL. For...

  15. Kit Free RNA Extraction

    Type
    Protocol
    ...tissues: use 1 mL of Solution D per 100 mg of cells. For cultured cells: use 1 mL of Solution D per 1 X 10 7...tissues: use 1 mL of TRIzol® per 100 mg of cells. For cultured cells: use 1 mL of TRIzol® per 1 X 10 7 cells... to 1 mL of lysate: Add 0.1 mL of 2 M sodium acetate (pH 4.0), mix thoroughly by inversion. Add 1 mL water-saturated...Option A): Add 1 volume of Isopropanol to the extracted aqueous layer. Incubate at -20°C for 1 hour. Lithium.... Add 0.2 mL of Chloroform/Isoamyl alcohol (49:1) per 1 mL of TRIzol® used. Shake vigorously by hand for... User Guide from ThermoFisher Scientific . Figure 1: A diagram of the different steps in RNA extraction...

  16. Personal Protective Equipment (PPE) for BSL-1 and BSL-2 Labs

    Type
    Protocol
    ...biosafety levels. This protocol provides information for both biosafety level 1 (BSL-1) and biosafety level 2 ...(PPE) for BSL-1 and BSL-2 labs. Protocols... Protocols Personal Protective Equipment (PPE) for BSL-1 and BSL-2 Labs...Labs Personal Protective Equipment (PPE) for BSL-1 and BSL-2 Labs Intro to the Lab Bench Check out more ...2 (BSL-2). The BSL-1 classification is for labs working with low-risk microbes posing little to no threat...all of the precautions needed in BSL-1, however there are additional precautions that lie beyond PPE. Video...always wear glasses/goggles in addition to the BSL-1 requirements. Conclusion Although simple, following...

  17. Lab Safety for Biosafety Levels One and Two

    Type
    Protocol
    ...biosafety levels. This protocol provides information for both biosafety level 1 (BSL-1) and biosafety level 2 ...level has different safety requirements. BSL-1 is designated for those working with microbes that don’t ...or Vibrio cholerae . BSL-2 includes all of the precautions needed in BSL-1, along with additional precautions...guidelines provide steps to ensure you are working in BSL-1 and BSL-2 labs safely. Protocols...Safety for Biosafety Levels One and Two (BSL-1 and BSL-2) Intro to the Lab Bench Check out more protocols...container Fire blanket Fire extinguisher Guidelines BSL-1 Guidelines Before You Work Right after entering the...hygiene officer. BSL-2 Guidelines Remember, the BSL-1 laboratory guidelines above are expected to be followed...

  18. AAV Production in HEK293 Cells

    Type
    Protocol
    ...7.5% Sodium Bicarbonate, 7.5 mL 1 M HEPES to 750 mL DMEM + 1 g/L glucose. 1 mg/mL polyethylenimine (PEI) ...Calculate the amount of each plasmid needed to have a 1:1:1 molar ratio with 2 mg total DNA per CS5 Plasmid ... determine the total μg/bp we need to achieve a 1:1:1 molar ratio of each plasmid: 2000 μg / 24,961 bp...deionized water + 100 mL of 1 M Tris HCl pH 8.5 + 60 mL of 5 M Sodium Chloride + 4 mL of 1 M Magnesium Chloride...high glucose, Corning 10-013-CV DMEM, low glucose (1 g/L) glucose, sodium pyruvate, Corning 10-014-CV (... sodium bicarbonate, Corning 25-035-CI (optional) 1 M HEPES, HyClone SH30237.01 (optional) L-alanyl-L-glutamine...cations can affect the attachment of adherent cells) 1 mg/mL Polyethylenimine (PEI) 25 kDa MW Pro-Tip Other...

  19. Antibody Validation Using the Indirect ELISA Method

    Type
    Protocol
    ...Data Figure 1: The plate was coated with serial dilutions of human recombinant purified Desmin (blue) or...avoid breathing in the vapors. Workflow Timeline Day 1: Antigen Coating Day 2: Blocking Day 3: Primary antibody... Spectrophotometer compatible with 96-well plates 1–10 µL single channel pipette 2–20 µL single channel... µL, VWR 76322-134 Pipettes, 10 mL, VWR 89130-898 1 L polystyrene bottle, Corning 430518 PBS, 1X pH 7.4...Warm reagents to room temperature. Procedure Section 1: Prepare the Antigen Standard and coat the plate Dilute...stock into 900 µL PBS in a microfuge tube and vortex. 1 ng/µL : Add 450 µL of 2 ng/µL stock into 450 uL PBS...microfuge tube and vortex. 0.5 ng/µL : Add 450 µL of 1 ng/µL stock into 450 µL PBS in a microfuge tube and...

  20. Ligation Independent Cloning

    Type
    Protocol
    ...DNA 10-50 ng/μl 1 dGTP (100mM) 2.5 mM 2 DTT (100 mM) 5 mM 1 BSA (10 μg/μl) 0.25 μg/μl 1 T4 DNA polymerase... treated vector and insert at a molar ratio of 1:2 or 1:3, using between 20 and 50 ng of vector per annealing...experimental design. Search Addgene's collection of empty LIC cloning vectors Protocol Step 1: Design Your Primers...reaction for 5 minutes at room temperature, then add 1 μl of 25 mM EDTA, followed by another 5 minutes at...The reaction is now ready for transformation. Use 1-2 μl of annealing reaction for each transformation... Primers Primer design for LIC is often as simple as using the backbone manufacturer's suggested leader...primer is shown here. Note: Use web-based primer design software to ensure a melting temperature between...

  21. Colony Formation Titering Assay

    Type
    Protocol
    ...Dilution 1:10 100 of Stock Virus 900 150 1,350 1:100 1:100 100 of 1:10 900 150 1,350 1:1,000 1:1,000 100...100 of 1:100 900 150 1,350 1:10,000 1:10,000 100 of 1:1,000 900 150 1,350 1:100,000 1:100,000 100 of 1... 1:10,000 900 150 1,350 1:1,000,000 Mix the dilutions well Note: the 1:10 dilution can usually be omitted...with 1 mL of 0.1% crystal violet for 10 min at RT. Gently remove the stain. Wash cells 3x with 1 mL of... media from the wells. Gently wash the cells with 1 mL of PBS and aspirate wash. Filter 0.1% crystal violet...volume in the well (mL) x dilution factor e.g., If the 1:100,000 well has 75 colonies, then there are 75 colonies...average of multiple dilutions. Sample Data Figure 1: A549 cells were transduced with the indicated serial...

  22. Isolating a Monoclonal Cell Population by Limiting Dilution

    Type
    Protocol
    ...Because this is such a small volume, first make 1 mL of a 1:100 dilution of the homogenized cell solution... were transduced with lentiCas9-Blast 1 and then selected with 1 μg/mL blasticidin for 9 days. Single ...with lentiCas9-Blast 1 . A549 cells were transduced (MOI = 37) and selected with 1 μg/mL blasticidin for...control. 1 lentiCas9-Blast was a gift from Feng Zhang (Addgene plasmid #52962 ) and is described in Improved...conditioned medium (see below for more details) Day 1: Seed individual cells in a 96-well plate Day 2–14...final 5 cell/mL solution, transfer 12.5 µL of the 1:100 dilution to 10 mL of conditioned medium. Transfer...cells will appear as colonies in the well ( Figure 1 ). You will be able to tell if there was more than...

  23. Virus Protocol - Generating Stable Cell Lines

    Type
    Protocol
    ...mL polybrene (µL) 0 0 500 1:5 300 200 1:10 150 350 1:50 30 470 1:100 15 485 1:500 3 497 Add 0.5 mL of a... if an MOI >1 was used, some cells may have 1 copy of the transgene, while others have >1 copy of the ... well by pipetting or inverting the tube. Aliquot 1 mL of cell suspension (i.e., 50,000 cells) into each... early polyclonal populations. Sample Data Figure 1: Generation of monoclonal cell lines from expansion...transduced with lentiCas9-Blast and then selected with 1 µg/mL blasticidin for 9 days. Single cells were ...with Lentivirus You may also like... Viral Vector Guides Virus Blog Posts Mol Bio Protocols Viral Service...dilutions and pick the population that has the most desirable level of expression. Over time, transgene expression...

  24. CRISPR Library Amplification

    Type
    Protocol
    ...Vented Falcon Tubes at 30-37 ℃, 225 rpm for 1 hour. After the 1 hour shaking period, pool and gently mix ...follow all specifications described in the equipment manual. Immediately add 1 mL SOC to cuvette. Remove...Last Update: August 17, 2023 Workflow Timeline Day 1: Transform, recover, set up overnight growth (Estimated... (Default: 4 tubes of Endura Duos, Lucigen, 60242-1) Alternatives include Stbl4 cells or other ultra-high...0.1 cm ) 20 mL SOC recovery media (Lucigen, 80026-1) 8X LB Agar + Antibiotic 245 mm bioassay plates (Molecular...into each of four 14 mL Vented Falcon Tubes and have 1 mL SOC per electroporation readily available for post-electroporation...autoclaved, sterile reagents for all steps. Procedure Day 1 Add 200 ng DNA to each 50 µL aliquot of thawed Endura...

  25. Lentivirus ddPCR Titration

    Type
    Protocol
    ...95 10 2 1 Denaturation 94 0.5 2 40 Annealing/Extension 60 1 2 40 Enzyme Deactivation 98 10 2 1 Hold 4 ... primers/probe (FAM) 1 µL 9 µL 900 nM, 250 nM 20X RPP30 primers/probe (HEX/VIC) 1 µL 9 µL 900 nM, 250 .... Last Update: July 7, 2023 Workflow Timeline Day 1: Seed and transduce cells Day 4: Treat cells with ...Cycler, Bio-Rad, T100 PCR Plate Sealer, Bio-Rad, PX1 1–10 µL single channel pipette 20–200 µL single channel...TrypLE, Thermo Fisher, 12605010) Ethanol, VWR, EX0276-1 Benzonase 250 U/µl, Millipore #71205-3 Polybrene 10... each viral dilution to a well of a 6-well plate (1 dilution per well). Leave one well untransduced (add...suspension well before seeding. Mix each well with a 1 mL pipette 5–10 times. The final volume in the well...

  26. AAV Titration by qPCR Using SYBR Green Technology

    Type
    Protocol
    ...dilution Dilution 1 (DNase step) 5 uL AAV stock 45 uL 10X 10X Dilution 2 5 uL Dil. 1 95 uL 20X 200X Dilution...range from 1 x 10 12 GC/mL to >2 x 10 13 GC/mL and we use an internal reference virus that is 1 x 10 13 ...Equipment qPCR instrument Heating plate Pipettors 1–10 µL single channel pipette 20–200 µL single channel...channel pipette 200–1000 µL single channel pipette 1–10 µL multichannel pipette 2–50 µL multichannel pipette...The reference material should have a titer within 1-log of the expected titer of the samples being tested...Amount for ONE Reaction Amount for 100 reactions (1 x 96 well plate) Universal SYBR Master Mix 2X 10 μL...mole)/bp = 4.03 x 10 6 g/mole Moles/μl: 1.07 μg/μL x 1 g/10 6 μg x1 / 4.03 x 10 6 g/mole = 2.65 x 10 -13 ...

  27. Transfection for Recombinant Antibodies

    Type
    Protocol
    ...from 1:1 to 1:6. Add the diluted PEI-MAX to the diluted DNA. Cap the tube and vortex with three 1 s pulses...solution at -20 °C. 1 mg/mL PEI-MAX Add 1 g of PEI-MAX powder to 900 mL deionized water in a 1 L bottle and ...to mix. Add 450 µL of 1 mg/mL PEI-MAX to the second tube of 6 mL BCD TFX (for a 1 mg/mL stock solution ...Last Update: February 18, 2022 Workflow Timeline Day 1: Seed cells Day 2: Transfect cells Day 3-6: Feed cells... a time. Add deionized water to a final volume of 1 L and recheck pH to ensure that it has not drifted...Aliquot and freeze upright at -20 °C. Procedure Section 1: Seeding cells The day prior to transfecting, seed...of two 50 mL tubes. Cap the tubes and incubate for 1 h in the 37 °C bead bath. Transfer the PEI-MAX and...

  28. Fluorescence Titering Assay

    Type
    Protocol
    ...polybrene (μL) 1:10 150 1348.5 1.5 1:25 60 1438.5 1.5 1:50 30 1468.5 1.5 1:75 20 1478.5 1.5 1:100 15 1483.5...the dilution factors (method 1) or the volume of virus (method 2): Method 1 Method 2 $$T = {N*F*D\over ...regulations. Workflow Timeline Day 0: Seed 293T cells Day 1: Transduce cells Day 2 (am): Remove media, replace...for 48–72 h. Gently aspirate media and replace with 1 mL of PBS. Calculate the fraction of fluorescent-positive... example: If 150,000 cells were transduced in the 1:100 well, resulting in 25% fluorescent cells, then...average of multiple dilutions. Sample Data Figure 1: 293T cells were transduced with a range of dilutions...for Lentivirus You may also like... Viral Vector Guides Virus Blog Posts Mol Bio Protocols Viral Service...

  29. Protocols for Molecular Biology, Plasmid Cloning, and Viral Preps

    Type
    Protocol
    ...(PPE) for BSL-1 and BSL-2 Labs Learn how to best protect yourself when working in BSL-1 and BSL-2 labs...Levels One and Two (BSL-1 and BSL-2) Safety measures for laboratories operating at BSL-1 and BSL-2 Watch the... T4 polymerase pLKO.1 - TRC Cloning Vector Cloning protocols for using the pLKO.1 vector, a backbone used...gel Watch the Video! How to Design a Primer Key considerations when designing primers Watch the Video! Sequence...Intro to the Lab Bench Introductory techniques designed to help you get started in the lab. Basic Molecular...antibody applications. Intro to the Lab Bench Name Description (Link opens in a new window) Link to Video Personal...materials on a balance Basic Molecular Biology Name Description (Link opens in a new window) Link to Video Making...

  30. Western Blot

    Type
    Protocol
    ...Prepare the chemiluminescence substrate by mixing 1:1 reagent A to reagent B. Gently incubate the membrane...Last Update: January 24, 2022 Workflow Timeline Day 1: Prepare lysates, run SDS-PAGE, transfer, block, incubate...primary antibodies raised in a mouse. Procedure Section 1: Lyse cells Centrifuge 5 x 10 6 cells for 5 min at...Gently remove supernatant. Resuspend cell pellet in 1 mL of 1X PBS and transfer to a microcentrifuge tube...preferred method for protein determination. Prepare a 50:1 Reagent A to Reagent B dilution of the BCA assay. ...side up. Block the membrane in blocking buffer for 1 h at RT on a shaking platform. Wash the membrane 3x... vary between antibodies but is typically between 1–10 μg/mL. Incubate the membrane overnight in primary...

  31. AAV Purification by Iodixanol Gradient Ultracentrifugation

    Type
    Protocol
    ...Preparation 1 M NaCl/PBS-MK buffer Dissolve 5.84 g of NaCl, 26.3 mg of MgCl 2 and 14.91 mg of KCl in 1× PBS ...gradient for AAV purification. Workflow Timeline Day 1: Purify Day 2: Buffer exchange and concentration Note...Dissolve 26.3 mg of MgCl 2 , and 14.91 mg of KCl in 1× PBS in a final volume of 100 mL. Sterilize by passing... step: mix 4.5 mL of 60% iodixanol and 13.5 mL of 1 M NaCl/PBS-MK buffer 25% iodixanol step: mix 5 mL ...Hamilton needle, taking care to avoid bubbles (Figure 1). 8 mL of 15% iodixanol step 5 mL of 25% iodixanol...disturb the gradient!** Collect Fractions Option #1 Prepare a row of roughly 20 open 1.5 mL microcentrifuge...with a 16 ga needle and start collecting 0.5 mL to 1 mL fractions per tube. Avoid the proteinaceous material...

  32. What is Polymerase Chain Reaction (PCR)

    Type
    Protocol
    ...annealing temperature step-wise by 1-2°C. The rate of DNA synthesis is ~1-2 kb/min. The extension time can...now bind to the primer DNA sequence. Extend DNA for 1 minute at 72°C: The Taq polymerase has an optimal ...DNA (10 ng-500 ng) 5 μl 10X Taq buffer with MgCl 2 1 μl dNTP mix (10 mM each nt) 2.5 μL Forward Primer ...primer melting temperature (Tm). Set extension step at 1-2 minutes per kilobase of product depending on whether...working concentration of each primer (10uM) by making a 1:10 dilution of the stock. For example, add 100µl of...Gel Procedure Primer Design and PCR Design Primers. See our protocol on how to design primers . Note: (Link...

  33. Coomassie Purity Stain of Recombinant Antibodies

    Type
    Protocol
    ...June 14, 2023 Workflow Timeline Day 1: Run SDS-PAGE and stain gel Day 1 or later: Image analysis Video Watch...lane Example for AR0018 (lane 2 in Figure 1): Sample Peak 1 (contaminant) Peak 2 (contaminant) Peak 3 ...antibodies using Coomassie stain. Equipment Heat block 1–10 µL single channel pipette 2–20 µL single channel...deionized water. Gently invert to mix. Procedure Section 1: SDS-PAGE Add 5 µL of 4X sample buffer to each sample...attach to a power supply. Run the gel at 150 V for 1 h . Pro-Tip If the samples are running unevenly and...Add 20 mL of SimplyBlue SafeStain and incubate for 1 h with gentle agitation on a rocking platform. Pour...sink. Add 100 mL of deionized water and incubate for 1 h with gentle agitation on a rocking platform. Pour...

  34. Immunocytochemistry

    Type
    Protocol
    ...Last Update: January 20, 2022 Workflow Timeline Day 1: Seed cells Day 3-4: Fix and label cells Equipment...into 5 mL PBS. Protect from light. Procedure Section 1: Seeding cells Place a sterile poly-D-lysine coated...concentration will vary but generally ranges from 1-10 µg/mL. Add 500 µL of the diluted antibody to the...concentration will vary but generally ranges from 1-10 µg/mL. Add 500 µL fluorescently-labeled secondary...with a laboratory wipe to remove excess liquid. Add 1 drop of anti-fade mounting medium to the microscope...This protocol describes the basic steps for fixing and staining cells in culture with a primary antibody...antibodies to detect antigens in cells. Here we describe the basic steps for fixing and labeling cells ...

  35. Protocol - Over-Agar Antibiotic Plating

    Type
    Protocol
    ... resistance) Procedure Day 1 Prepare carbenicillin to a concentration of 1 mg/mL – 4 mg/mL in LB medium...lawn of E. coli and no apparent selection. 150 µL of 1 mg/mL Carbenicillin plated over-agar Plate shows several...over-agar Plate shows less individual colonies than the 1 mg/mL plate and effective selection. 150 µL of 4 mg...several individual colonies with smaller size than the 1 mg/mL and 2 mg/mL plates and effective selection. ... Bacterial Culture Introduction This protocol describes methodology for plating antibiotic over-agar for...

  36. Protocol - Bacterial Transformation

    Type
    Protocol
    ...get more colonies if you use 1 μl of a 1:5 or 1:10 dilution rather than 1 μl directly....and then (optional) incubate in 37°C incubator. Mix 1 - 5 μl of DNA (usually 10 pg - 100 ng) into 20-50 ...shock each transformation tube by placing the bottom 1/2 to 2/3 of the tube into a 42°C water bath for 30...Because of this, nearly all plasmids (even those designed for mammalian cell expression) carry both a bacterial...

  37. Protocol - How to Ligate Plasmid DNA

    Type
    Protocol
    ...situations where the 3:1 ratio is not working or when doing more complicated cloning. While 3:1 will get you in...the insert is smaller than the vector) a 3 insert : 1 vector molar ratio will work just fine. We recommend...reagents Optimizing the Vector:Insert Ratio Although a 3:1 insert to vector ratio is usually sufficient, you ...phosphodiester linkages, which permanently join the nucleotides together. After ligation, the insert DNA is physically... likely to ligate to itself rather than to the desired insert. If you are in this situation, it is important...reactions. Because ligase buffer contains ATP, which degrades upon freeze/thaw cycles, it is a good idea to ...

  38. Protocol - How to Run an Agarose Gel

    Type
    Protocol
    ...Biolabs B7022S Procedure Pouring a Standard 1% Agarose Gel: Measure 1 g of agarose. Pro-Tip Agarose gels are.... Note: Loading buffer serves two purposes: 1) it provides a visible dye that helps with gel loading and...different buffers and do not use water). Microwave for 1-3 min until the agarose is completely dissolved (but...the way down the gel. A typical run time is about 1-1.5 hours, depending on the gel concentration and ...be pushed away from the well comb or towards the sides/edges of the gel with a pipette tip. Place newly...Always Run to Red. Turn OFF power, disconnect the electrodes from the power source, and then carefully remove...-30 mins, replace EtBr solution with water and destain for 5 mins. Using any device that has UV light,...

  39. Pouring LB Agar Plates

    Type
    Protocol
    ... chloride 12.0 g agar-agar 1 L Sterile H 2 O Sterile plates of your desired size - we usually use 60 mm...bottle for autoclaving. We make 400 mL of agar in 1 L bottles and 200 mL of agar in 500 mL bottles. The...measure out 100 mg of ampicillin powder, add it to 1 mL of water, dissolve by vortexing, and filter sterilize... You should not store your plates for longer than 1 month at any temperature and should always check for... for contamination prior to use. Negative Result 1: Both Strains Grow Assuming the appropriate strains...next to the flame and begin pouring. Measure your desired amount of agar with a pipete for the first plate...

  40. Plasmid Cloning by PCR (with Protocols)

    Type
    Protocol
    ...has error rates that range from roughly 1 per 500bp to roughly 1 per 10 million bp depending on the polymerase...recipient plasmid to insert ratio of approximately 1:3. Since the number of base pairs for each varies,...cells. For most standard cloning, you can transform 1-2μl of your ligation reaction into competent cells...plasmid of interest. For this example, we will describe how to copy a cDNA from one vector into a new ...(YGOI) for ligation into a recipient plasmid. Designing Primers for PCR Based Cloning The basic PCR primers...that: Do not cut within your insert. Are in the desired location in your recipient plasmid (usually in ...examine the DNA sequence that we want to amplify and design primers that will bind to and replicate it. The...

  41. Pipetting Protocol

    Type
    Protocol
    ...dispense small amounts of liquid (think: 0.1 µL to 1 mL). When working in a laboratory, properly dispensing...right. Pipette Dispense Volume P2 0.2 to 2 µL P10 1 to 10 µL P20 2 to 20 µL P100 10 to 100 µL P200 20 ...and the third (red) digit is hundredths. Therefore, 1 µL would read as 100 (as shown in the picture above...maximum possible setting on the pipette, set the desired volume of the liquid on the pipette. Depending ...of the pipette. Select the pipette tip that is designed to fit your pipette. To load a tip onto a pipette...from the container making sure not to touch the sides of the container with the pipette. Discard the pipette...

  42. Plasmid Modification by Annealed Oligo Cloning (with Protocols)

    Type
    Protocol
    ...annealing can be achieved by one of two methods: Method #1 Place the mixed oligos in a 1.5mL microfuge tube. ...vector in molar ratios (vector:insert) between 4:3 and 1:6 in a standard ligation reaction (ex. to ligate an...oligo overlap cloning, you can design a set of oligos containing your desired restriction sites and add them...that will pay off for years to come. Design Briefly, we will design overlapping oligos that once annealed...procedure will simply differ in terms of primer design). Let's assume that your favorite vector has a ...digest of existing sites in the original vector. Designing oligos To add NdeI, PacI, AscI and MfeI sites ...between the EcoRI and SalI sites of the vector, we design a top oligo with each of the additional sites in...

  43. Using a Light Microscope Protocol

    Type
    Protocol
    ...Figure 1 depicts an image of a compound light microscope with the main components labeled: Figure 1: Diagram...magnifies it 10 times, and so on. The ocular lens also provides magnification and the power should be provided...provided on the microscope; often this lens provides 10x magnification. To determine the final magnification...that object! Equipment Light Microscope Prepared Slides (or other sample that can fit on a microscope stage...eyepiece so that you can see your image through both sides. Once you are happy with the lighting, use the coarse...

  44. Plasmid Cloning by Restriction Enzyme Digest (with Protocols)

    Type
    Protocol
    ...recipient plasmid to insert ratio of approximately 1:3. Since the number of base pairs for each varies,...cells. For most standard cloning, you can transform 1-2μl of your ligation reaction into competent cells...restriction enzyme digest (subcloning), including design and experimental procedures. Protocols...easily move YGOI into a mammalian expression vector. Design (Choosing enzymes) Many DNA analysis tools, including..., but do not cut within your insert Are in the desired location in your recipient plasmid (usually in ...your recipient plasmid as well as a specifically designed test digest later to verify that the insert was... fear. You have other options, such as: Adding desired restriction sites to flank your insert : You can...

  45. DNA Quantification

    Type
    Protocol
    ... measure your samples, place 1-2µL of mini-prepped DNA onto the pedestal. Close the lid and click measure...Repeat for each sample. Notes: Keep in mind that despite the accuracy of the NanoDrop, if two consecutive...

  46. Water Bath Protocol

    Type
    Protocol
    ...quicker. Pro-Tip Set up the water bath 30 minutes to 1 hour before you need to use it to allow the temperature... of bacteria or fungi. There are disinfectants designed to be used in water baths with instructions on...bath on and set the appropriate temperature as described in your protocol. How to set the temperature will...Set up your water bath so that it will reach the desired temperature by the time you need it for your experiment...water bath to prevent evaporation and maintain the desired temperature. This also helps the water bath heat...water bath even if you are using disinfectants as described above in step 3. You will also need to maintain...

  47. Gibson Assembly Protocol

    Type
    Protocol
    ...ssDNA. (Rabe & Cepko, 2020). Incubate the mix for 1 hour at 50 °C or follow manufacturer's instructions... Procedure Design your plasmid and order primers (see figure to the right). When designing your plasmid...and enrich for correctly assembled plasmids by designing primers to split an antibiotic resistance gene...and yield. If there are significant amounts of undesired product, gel-purify DNA segments. Otherwise, PCR... High-Fidelity DNA Polymerase - incorporates nucleotides to “fill in” the gaps in the annealed DNA fragments... (<150 bp). Pro-Tip Please note that the way to design the “stitching” primers and the amounts of primers...window) . NEB has other resources, such as a primer design tool (Link opens in a new window) . Gibson Assembly...

  48. Handling Plasmids from Addgene - Purifying Plasmid DNA

    Type
    Protocol
    ...solution, add 2-2.5 volumes 95% or 100% ethanol and 1/10 volume of 3 M Na-acetate (pH 4.8). Invert the microfuge...Last Update: Feb. 8, 2018 Equipment Desktop microcentrifuge Desktop vortexer Vacuum (optional) Reagents... several μg/μL. The protocol below is meant to describe the general procedure for purifying plasmid DNA...
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