We narrowed to 29 results for: MLS

1. AAV Purification by Iodixanol Gradient Ultracentrifugation

   Type

   Protocol

   ...Figure 1). 8 mL of 15% iodixanol step 5 mL of 25% iodixanol step 5 mL of 40% iodixanol step 3 mL of 60% iodixanol...: 49.5 mL PBS + 500 µl Pluronic F68 B: 0.01% in PBS: 45 mL PBS + 5 ml A C: 0.001% in PBS: 45 mL PBS + ...Pluronic-F68 to 49.5 mL PBS 0.01% Pluronic-F68 in PBS (B) Add 5 mL of Buffer A to 45 mL PBS 0.001% Pluronic-F68...) (formulation buffer) Add 5 mL of Buffer B and 2 mL of 5 M NaCl to 43 mL PBS Procedure Preparation and...mix 4.5 mL of 60% iodixanol and 13.5 mL of 1 M NaCl/PBS-MK buffer 25% iodixanol step: mix 5 mL of 60% ...iodixanol and 7 mL of 1X PBS-MK buffer and 30 μL of phenol red 40% iodixanol step: mix 6.7 mL of 60% iodixanol...iodixanol and 3.3 mL of 1X PBS-MK buffer 60% iodixanol step: mix 10 mL of 60% iodixanol and 45 μL of phenol...

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

   Type

   Protocol

   ...265 5 mL pipette, VWR 89130-896 10 mL pipette, VWR 89130-898 25 mL pipette, VWR 89130-900 50 mL pipette... T2819-1L 250 mL sterile bottle, VWR 430281 0.45 µm PES complete filtration unit, 500 mL, rapid-flow, ..., 10 mL, Thermo Fisher 89893 Amicon Ultra-15 30 kDa MWCO column, Millipore Sigma UFC903024 50 mL LoBind...pH 7.0 390 mL of sterile sodium phosphate monobasic monohydrate (NaH 2 PO 4 ∙H 2 O) 610 mL of sterile ...sodium phosphate buffer, pH 7.0) 20 mL 1M sodium phosphate buffer 980 mL of deionized water Adjust pH to ...leupeptin 50 mg antipain 250 mg benzamidine 25 mL of 2 mg/mL aprotinin Mix well and sterilize through a 0.2...column with 10 mL of RT Protein A/G binding buffer. re Collect the flow-through in a 50 mL conical tube ...

3. Transfection for Recombinant Antibodies

   Type

   Protocol

   ... 500 mL vented flask 50 mL conical tubes Microcentrifuge tubes Cell counting chamber slide 30 mL luer-... until use. BCD TFX 1000 mL BalanCD HEK293 Media 10 mL 10% Pluronic-F68 40 mL 200 mM Glutagro Do not add...transfecting, seed a 108 mL culture of HEK293 cells at a density of 0.9 x 10 6 cells/mL in a 500 mL vented flask....PES filtering system, 1000 mL, VWR 431098 0.45 µm PES filtering system, 500 mL, VWR 430770 Trypan Blue, ...in 200 mL deionized water Sterilize by passage through a sterile 0.22 µm filter. Prepare 10 mL aliquots...the 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...solutions after one month. BCD Feed 500 mL BalanCD HEK293 Feed 20 mL 200 mM Glutagro Store at 4 °C until ...

4. AAV Production in HEK293 Cells

   Type

   Protocol

   ... 500 mL bottle of DMEM high glucose, add 55 mL of heat-inactivated FBS and 5 mL of glutaGRO 11 mL of 200...sorbitol, add 57 mL of 1.46 M sorbitol, 7.5 mL HI-FBS, and 7.5 mL 100X glutaGRO, 7.5 mL of 7.5% Sodium ...: 1836 mL deionized water + 100 mL of 1 M Tris HCl pH 8.5 + 60 mL of 5 M Sodium Chloride + 4 mL of 1 M...Sodium Bicarbonate, 7.5 mL 1 M HEPES to 750 mL DMEM + 1 g/L glucose. 1 mg/mL polyethylenimine (PEI) solution... 468.1 μL Aliquot 100 mL of OptiMEM into a sterile 250 mL bottle. Aliquot 774 mL of DMEM + 2% HI-FBS into... is 8%. Add 25 mL of PEG solution to each 100 mL of supernatant. Split into 2 x 500 mL sterile bottles...culture media and rinse once with 10 mL of PBS. Aspirate PBS and add 2 mL of 0.05% Trypsin/EDTA. Wait ~2 min...

5. Pouring LB Agar Plates

   Type

   Protocol

   ... mg/mL 10 µg/mL Kanamycin 50 mg/mL 50 µg/mL Spectinomycin 50 mg/mL 50 µg/mL Tetracycline 10 mg/mL 10 µg...Ampicillin 100 mg/mL 100 µg/mL Bleocin 5 mg/mL 5 µg/mL Carbenicillin* 100 mg/mL 100 µg/mL Chloramphenicol...Chloramphenicol 25 mg/mL (dissolve in EtOH) 25 µg/mL Coumermycin 25 mg/mL (dissolve in DMSO) 25 µg/mL Gentamycin 10... 10 mL, we’ll want 200 mL of media total. Importantly, we’ll actually make a bit more than 200 mL (~220...autoclaving. We make 400 mL of agar in 1 L bottles and 200 mL of agar in 500 mL bottles. The extra empty...concentration of 100 ug/mL ampicillin, you should make a stock solution of 100,000 ug/mL (100 mg/mL). Simply measure...usually use 60 mm x 15 mm plates which can hold 5-10 mL of agar and on which you can individually distinguish...

6. Fluorescence Titering Assay

   Type

   Protocol

   ...glutaGRO) To a 500 mL bottle of DMEM high glucose, add 55 mL of heat-inactivated FBS and 5 mL of 100X glutaGRO...fluorescent x 100)/(1.5 mL) = 2.5x10 6 TU/mL $$T = {N*F\over V_V}$$ Where: T = Titer, TU/mL N = Number of cells...525,000 cells into 14 mL of DMEM complete. Mix well by pipetting or inverting. Aliquot 2 mL of cell suspension... μg/mL polybrene. Note, this protocol was developed using low titer lentiviral vectors (10 5 TU/mL). If...true titer. Calculate the transduction units per mL (TU/mL) using either the dilution factors (method 1) ...150,000 cells x 0.25 fluorescent)/(0.015 mL) = 2.5x10 6 TU/mL For a more accurate titer, take the average...Corning 25-015-CI) Heat-inactivated FBS Polybrene (10mg/mL), Millipore TR-1003-G 1X PBS pH 7.4 without calcium...

7. Coomassie Purity Stain of Recombinant Antibodies

   Type

   Protocol

   ...1.0 mg/mL: 20 µL of 1.5 mg/mL with 10 µL PBS 0.75 mg/mL: 15 µL of 1.0 mg/mL with 5 µL PBS 0.5 mg/mL: 13.3...µL of 0.75 mg/mL with 6.7 µL PBS 0.25 mg/mL: 10 µL of 0.5 mg/mL with 10 µL PBS 0.125 mg/mL: 10 µL of 0.25...76322-154 Pipettes, 5 mL, VWR 89130-896 Pipettes, 10 mL, VWR 89130-898 Pipettes, 25 mL, VWR 89130-900 Pipettes...Dilute the 2.5 mg/mL IgG isotype standard in PBS as follows: 1.5 mg/mL: 18 µL of 2.5 mg/mL with 12 µL PBS...45000-446 250 mL sterile bottles, Corning 430281 Deionized water IgG isotype standard 2.5 mg/mL - can use ... Prepare 1X MOPS buffer Dilute 25 mL of 20X MOPS buffer into 475 mL of deionized water. Gently invert ...Reagents Purified recombinant antibody, 0.9–1.1 mg/mL SimplyBlue SafeStain, Thermo Fisher, LC6060 4-12%...

8. Colony Formation Titering Assay

   Type

   Protocol

   ...transduction units per mL (TU/mL) as follows: TU/mL = number of colonies/total volume in the well (mL) x dilution...glutaGRO) To a 500 mL bottle of DMEM high glucose, add 55 mL of heat inactivated FBS and 5 mL of 100X glutaGRO...complete containing 10 μg/mL polybrene by diluting 20 μL of 10 mg/mL polybrene into 20 mL of media. If using ...: Dilute 7,000 cells into 9.45 mL of DMEM complete containing 10 μg/mL polybrene. Mix well by pipetting...with 1 mL of 0.1% crystal violet for 10 min at RT. Gently remove the stain. Wash cells 3x with 1 mL of PBS..., then there are 75 colonies /1.5 mL x 1,000,000 or 50,000,000 TU/mL For a more accurate titer, take the... 25-015-CI) Heat-inactivated FBS Polybrene (10 mg/mL), Millipore TR-1003-G 1X PBS pH 7.4 without calcium...

9. CRISPR Library Amplification

   Type

   Protocol

   ... of the four 14 mL Vented Falcon Tubes should contain a total of 5 mL (3 mL SOC + 2 mL transformed Endura...freezer. Aliquot 3 mL SOC into each of four 14 mL Vented Falcon Tubes and have 1 mL SOC per electroporation...slightly. We routinely use 350 mL of LB Agar per bioassay plate. 14 mL Vented Falcon Tubes (BD Biosciences... µL) Bacti Cell Spreaders (VWR, 60828-680) 5 mL and 10 mL Serological pipettes Ice slurry (Ice bucket ...Immediately add 1 mL SOC to cuvette. Remove all liquid from cuvette and add to 14 mL vented Falcon Tube...electroporation cuvettes (BioRad, Micropulser, 0.1 cm ) 20 mL SOC recovery media (Lucigen, 80026-1) 8X LB Agar ...bucket with ice and water to create slurry) 100 mL LB At Addgene we use premixed LB (VWR 101414-072) for convenience...

10. Virus Protocol - Generating Stable Cell Lines

    Type

    Protocol

    ...DMEM complete + 10 µg/mL polybrene by diluting 20 µL of 10 mg/mL polybrene into 20 mL media. Thaw the lentiviral...alternative, such as glutaGRO) To a 500 mL bottle of DMEM high glucose, add 55 mL of heat-inactivated FBS and 5mL...,000 cells into a total volume of 7 mL of DMEM complete + 10 µg/mL polybrene. Mix well by pipetting or...well up to 1.5 mL. Since all the media in these wells was made with DMEM complete + 10 µg/mL polybrene, the... 25-015-CI) Heat-inactivated FBS Polybrene (10 mg/mL), Millipore TR-1003-G 1X PBS pH 7.4 without calcium...well tissue culture treated dish, Corning 3516 15 mL conical tubes, VWR 21008-216 Lentivirus preparation...dilutions of the lentivirus in DMEM complete + 10 µg/mL polybrene. Note, this is just a sample of possible...

11. Protocol - How to Inoculate a Bacterial Culture

    Type

    Protocol

    ...100 µg/mL Bleocin 5 µg/mL Carbenicillin 100 µg/mL Chloramphenicol 25 µg/mL Coumermycin 25 µg/mL Gentamycin...Gentamycin 10 µg/mL Kanamycin 50 µg/mL Spectinomycin 50 µg/mL Tetracycline 10 µg/mL Tips and FAQ What is the.... For example, to make 400 mL of LB, weigh out the following into a 500 mL glass bottle: 4 g NaCl 4 g ...g Tryptone 2 g Yeast Extract and dH 2 O to 400 mL Note: If your lab has pre-mixed LB agar powder, use ...to do a mini-prep you will usually want to start 2 mL in a falcon tube, but for larger preps you might ...

12. General Transfection

    Type

    Protocol

    ...glutaGRO) To a 500 mL bottle of DMEM high glucose, add 55 mL of heat-inactivated FBS and 5 mL of 100X glutaGRO...months. 1 mg/mL polyethylenimine, linear MW 25,000 Da (PEI) Dissolve 100 mg of powder into 100 mL of deionized... a volume of 15 mL. Wednesday: Plate 1x10 6 cells in a T75 flask in a volume of 15 mL. Friday: Plate 8x10...add 10 mL fresh DMEM complete containing 25 µM chloroquine diphosphate and incubate ~5 h For 10 mL of DMEM...Dissolve 0.129 g of chloroquine diphosphate salt into 10 mL of sterile water. Filter sterilize through a 0.22... 8x10 5 cells in a T75 flask in a volume of 15 mL. Do not add antibiotics to the media. Use cells that...empirically determined for each new batch of 1 mg/mL PEI prepared. There may be variation between batches...

13. Isolating a Monoclonal Cell Population by Limiting Dilution

    Type

    Protocol

    ...glutaGRO) To a 500 mL bottle of DMEM high glucose, add 55 mL of heat-inactivated FBS and 5 mL of 100X glutaGRO...concentration: 4 × 10 5 cells/mL To seed one 96-well plate, make 10 mL of a 5 cell/mL solution. Calculate the...solution at a concentration of 5 cells/mL. Prepare approximately 10 mL of this cell solution for each 96-well...total cells needed: Total cells needed: 10 mL × 5 cells/mL = 50 cells Determine the volume of homogenized.../(4 × 10 5 cells/mL) = 0.125 µL Because this is such a small volume, first make 1 mL of a 1:100 dilution...make the final 5 cell/mL solution, transfer 12.5 µL of the 1:100 dilution to 10 mL of conditioned medium...10 cm dish. Each 10 cm dish should be seeded in 10 mL DMEM complete, which will generate enough conditioned...

14. Protocol - Over-Agar Antibiotic Plating

    Type

    Protocol

    ...Prepare carbenicillin to a concentration of 1 mg/mL – 4 mg/mL in LB medium. The concentration of antibiotic...of 2 mg/mL Carbenicillin plated over-agar Plate shows less individual colonies than the 1 mg/mL plate and...individual colonies with smaller size than the 1 mg/mL and 2 mg/mL plates and effective selection. Selection Curve...plates without antibiotic High concentration (100 mg/mL, 1000x) carbenicillin stock solution in sterile water...lawn of E. coli and no selection. 150 µL of 0.1 mg/mL Carbenicillin plated over-agar Plate shows a lawn...E. coli and no apparent selection. 150 µL of 1 mg/mL Carbenicillin plated over-agar Plate shows several... and effective selection. 150 µL of 4 mg/mL Carbenicillin plated over-agar Plate shows several individual...

15. Lentivirus Production

    Type

    Protocol

    ...glutaGRO) To a 500 mL bottle of DMEM high glucose, add 55 mL of heat-inactivated FBS and 5 mL of glutaGRO. ...min. 1 mg/mL polyethylenimine, linear MW 25,000 Da (PEI) Dissolve 100 mg of powder in 100 mL of deionized...T75 flask in 15 mL DMEM Complete. Wednesday: Plate 1×10 6 cells in a T75 flask in 15 mL DMEM Complete. ...at RT. During the incubation, add 10 mL of DMEM Complete to a 15 mL conical and an appropriate amount of...3745.X 10 cm tissue culture dish, Corning 430167 15 mL conical tubes, VWR 21008-216 Hydrochloric acid Sodium...empirically determined for each new batch of 1 mg/mL PEI and for each cell line. Considerations Before.... Friday: Plate 8×10 5 cells in a T75 flask in 15 mL DMEM Complete. Do not add pen-strep to the media....

16. Lentivirus ddPCR Titration

    Type

    Protocol

    ...500 mL DMEM, high glucose, pyruvate 55 mL heat inactivated premium grade, fetal bovine serum 5 mL glutaGRO...glutaGRO 50 U/mL benzonase: 15 mL DMEM Complete 3 µL of 250 U/µL benzonase Procedure Transducing Cells Thaw...10 wells with 3,000,000 cells in 13.5 mL of media and 11.1 µg/mL Polybrene. Mix the cell suspension well... each well with a 1 mL pipette 5–10 times. The final volume in the well is 1.5 mL, and the final Polybrene...cell conditions with others. Gently add 1.5 mL of 50 U/mL Benzonase in DMEM complete to each well. Incubate...titer, TU/mL V = Viruses per genome C = # Cells per well D = Dilution factor v = Virus volume, mL *In the...protocol is based on an LV titer range of 1E+05–1E+09 TU/mL, where TU is transducing units and generally represents...

17. Immunocytochemistry

    Type

    Protocol

    ...Anti-fade mounting medium Laboratory wipes 15 mL conical tubes 50 mL conical tubes Before Starting Refer to ...Triton X-100 in 10 mL PBS. Blocking buffer: Dilute 0.5 g BSA and 30 µL Triton X-100 in 10 mL PBS. Antibody ...HeLa cells 24-well plate 4% Paraformaldehyde 5 mg/mL 4′,6-diamidino-2-phenylindole (DAPI) Bovine serum...buffer: Dilute 0.5 g BSA and 150 µL Triton X-100 in 50 mL PBS. 300 nM DAPI working solution: Prepare a 300 ...DAPI stock solution by diluting 2.1 µL of the 5 mg/mL DAPI solution to 100 µL PBS. Protect from light. ...diluting 5 µL of the 300 µM DAPI stock solution into 5 mL PBS. Protect from light. Procedure Section 1: Seeding...concentration will vary but generally ranges from 1-10 µg/mL. Add 500 µL of the diluted antibody to the wells ...

18. Kit Free RNA Extraction

    Type

    Protocol

    ...cell lysate to a 4 mL tube. Add the following sequentially to 1 mL of lysate: Add 0.1 mL of 2 M sodium acetate...For tissues: use 1 mL of Solution D per 100 mg of cells. For cultured cells: use 1 mL of Solution D per... inversion. Add 1 mL water-saturated phenol, mix thoroughly by inversion. Add 0.2 mL of Chloroform/Isoamyl...For tissues: use 1 mL of TRIzol® per 100 mg of cells. For cultured cells: use 1 mL of TRIzol® per 1 X ...homogenized sample(s). Add 0.2 mL of Chloroform/Isoamyl alcohol (49:1) per 1 mL of TRIzol® used. Shake vigorously...solution RNase free tubes: microcentrifuge tubes, 4 mL polypropylene tubes RNase decontamination solution... Wash the RNA by resuspending the pellet in 0.5–1 ml of 75% Ethanol and vortex for a few seconds. Centrifuge...

19. Western Blot

    Type

    Protocol

    ...Prepare 1X TBST as follows: 25 mL of 20X TBS 2.5 mL of Tween-20 472.5 mL of deionized water Mix well Prepare...running buffer as follows: Dilute 25 mL of 20X running buffer to 500 mL with deionized water. Mix well. Fill...water. To prepare 20% ethanol, dilute 2 mL of ethanol into 8 mL of deionized water and mix well. Assemble...Gently remove supernatant. Resuspend cell pellet in 1 mL of 1X PBS and transfer to a microcentrifuge tube....dilutions of the BSA standard that range from 0–2000 µg/mL . In duplicate, dilute 10 µL of standard, blank, ...into 1X TBST Example: 5 g of non-fat milk into 100 mL of 1X TBST. Pro-Tip The ideal blocking buffer will...between antibodies but is typically between 1–10 μg/mL. Incubate the membrane overnight in primary antibody...

20. Handling Plasmids from Addgene - Purifying Plasmid DNA

    Type

    Protocol

    ...Potassium Acetate) 120 mL 5M Potassium acetate 23 mL glacial acetic acid 57 mL of dH 2 O Store Solution... of interest. Add 1.5 mL of the stock culture to a 1.75 mL microfuge tube. Centrifuge in microfuge tube...buffer Denaturing solution Renaturing solution 2 mg/mL RNase A TE or water-saturated phenol-chloroform Chloroform...Solution III at 4°C Grow 2 mL overnight cultures from single colonies of bacteria containing your plasmid of... or carefully pouring. Optional: Add 5 μL of 2 mg/mL RNase A to the supernatant in the new tube and incubate...