karyotyping Practical Procedure
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This presentation includes the basic knowledge of Karyotyping with a lot of understandable knowledge and also how to use it properly. I hope all the finders liked it and also remember me in your precious Dua. Thank You!
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![14. Remove most of sup, and resuspend pellet in remaining media.
15.Using a dropper, slowly add 10 drops of Carnoyʼs fixative, flicking tube in
between drops to prevent clumping. Bring volume to 2ml with fixative.
16.Spin 5 minutes.
17. Remove supernatant and slowly add 4ml of fixative, flick tube, spin and
discard sup. Repeat, but donʼt remove all of sup. the second time around.
Leave about 1ml of sup. and flick.
18.When you are ready to drop the cells, fill plastic box with ice and pour
enough of the chilled ddH20 and Ethanol into two separate beakers to dip
and cover the cold slides. Leave the beakers on ice. (Use slide holder for
ethanol and large square beaker for ddH20).
19.Dip slides in chilled 70% ethanol and then rinse well with in the chilled
ddH2O. Leave slides in the chilled water until you are ready to use them –
slides must remain COLD in order for karyotype to work!!!. Using a glass
pipette, aspire some of the cells and drop them onto the clean, wet and
chilled slides either at arms length away or at varying heights from the
floor. Dry slides on hot plate at LOW setting.
20.Stain with filtered Giemsa for 4 minutes.
21.Wash with 1x PBS for 5 minutes.
22.Wash with ddH2O.
23.Count at least 15 cells. Significant problem if more than 4 cells have more
or less than 40 chromosomes (mouse) [or 46 for human].
Solutions for chromosome preparation
Vinblastine sulfate salt. Sigma, cat# V1377
Add 0.5 ml sterile water to original vial containing 1mg of crystal vinblastine
sulfate for a 2mg/ml stock solution. Aliquot and dilute to working solution of
25ul/25mlsterile water. Keep stock and working solution covered with foil at 4C.
Hypotonic solution
1.5g sodium citrate
1.12 g potassium chloride
q.s to .5L](data:image/gif;base64,R0lGODlhAQABAIAAAAAAAP///yH5BAEAAAAALAAAAAABAAEAAAIBRAA7)
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Objectives
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· Use aseptic technique during set up and inoculation
· Select 3 tubes of broth if using 2ml: Label two ara/amp and one amp. If using 5 ml tubes of LB broth use 2 tubes label one ara/amp and one amp.
· Add (9ul /1ml) each of arabinose and ampicillin to two tubes (ara/amp)
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· Place tubes in a rack and incubate in a shaker overnight at 32oC. Be sure to shake vigorously.
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RNA extraction, its purpose, methods, gel electrophoresis its types, agarose and polyacrylamide gel electrophoresis.
Biotechnology experiments 2nd semester (LNMU Darbhanga)
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Zearalenone
This document provides instructions for using an ELISA kit to detect the mycotoxin zearalenone in cereal crops and animal feeds. It begins with an introduction to zearalenone and its health effects. It then describes the intended use, principle, reagents, materials, precautions, extraction procedure, and assay procedure for the zearalenone ELISA kit. The kit is designed to quantitatively detect zearalenone in samples through a competitive enzyme immunoassay.
MTT Assay (cell spiltting + cell counting)
The MTT assay is a colourimetric assay for assessing cell metabolic activity. This slideshare includes complete steps with real images showing all steps starting from cell spiltting, cell counting and then MTT Assay.
Take a brief idea from this slideshare and try to do it by yourself in your respective labs in order to get more idea about MTT Assay.
NGUYENTHINHI-W3.pptx
Nguyen Thi Nhi completed the following works in the third week of her master's program:
1) Learned techniques for MIN6 cell culture including making media and using an autoclave.
2) Studied cell culture and immunoassay techniques in theory.
3) Specifically learned how to make cell culture media including DMEM, FBS, penicillin/streptomycin, and beta-mercaptoethanol and the purpose of each component. She also learned proper operation of the JSR autoclave for sterilization.
DNA extraction method for Plant sample
The CTAB method is used to extract DNA from plant cells. It involves lysing plant cells with the cationic detergent CTAB in a similar way that SDS is used to lyse bacterial cells. The procedure freezes and grinds a plant sample, incubates it in CTAB buffer, precipitates the DNA with chloroform, isoamyl alcohol and isopropanol, washes it with ethanol, and dissolves the final DNA pellet in TE buffer. Gel electrophoresis is then used to check the integrity of the extracted DNA.
Cell based antiviral assays for screening and profiling of Anti dengue agents
1. The document discusses protocols for culturing dengue virus, including culturing the virus in Vero cells and harvesting the viral supernatant.
2. It also describes methods for evaluating potential dengue virus inhibitors, including a plaque reduction assay using BHK-21 cells and a high-throughput antiviral screening assay measuring ATP levels in Vero cells.
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Quickly · SlidesCarnival presentation by mansi.pptx
This document provides instructions for several microbiology laboratory techniques and experiments. It includes procedures for preparing common microbiology media like nutrient broth, nutrient agar, and YPD agar. It also describes how to conduct experiments like methylene blue degradation, which is a photocatalytic reaction, and a protein cross-linking method using transglutaminase. The document provides detailed steps for culturing E. coli, using equipment like autoclaves, laminar flow hoods, and micropipettes, and following sterilization and contamination prevention protocols in the laboratory.
Total aflatoxin
This document provides instructions for performing an enzyme-linked immunosorbent assay (ELISA) to quantitatively detect total aflatoxins (B1, B2, G1, and G2) in grains, nuts, and other commodities. Aflatoxins are toxic metabolites produced by certain molds that can contaminate foods and animal feeds. The ELISA uses an aflatoxin-specific antibody to competitively bind sample aflatoxins or HRP-conjugated aflatoxin. The intensity of the color reaction indicates the aflatoxin concentration in samples, which can be quantified by comparison to standard curves. Validation studies showed the assay can reliably detect aflatoxins from 1-20 p
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Cell based antiviral assays for screening and profiling of Anti dengue agents
Cell based antiviral assays for screening and profiling of Anti dengue agents
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Quickly · SlidesCarnival presentation by mansi.pptx
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Description:
Dive into the fascinating realm of solid-state physics with our meticulously crafted online PowerPoint presentation. This immersive educational resource offers a comprehensive exploration of the fundamental concepts, theories, and applications within the realm of solid-state physics.
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Current descriptions of immersive learning cases are often difficult or impossible to compare. This is due to a myriad of different options on what details to include, which aspects are relevant, and on the descriptive approaches employed. Also, these aspects often combine very specific details with more general guidelines or indicate intents and rationales without clarifying their implementation. In this paper we provide a method to describe immersive learning cases that is structured to enable comparisons, yet flexible enough to allow researchers and practitioners to decide which aspects to include. This method leverages a taxonomy that classifies educational aspects at three levels (uses, practices, and strategies) and then utilizes two frameworks, the Immersive Learning Brain and the Immersion Cube, to enable a structured description and interpretation of immersive learning cases. The method is then demonstrated on a published immersive learning case on training for wind turbine maintenance using virtual reality. Applying the method results in a structured artifact, the Immersive Learning Case Sheet, that tags the case with its proximal uses, practices, and strategies, and refines the free text case description to ensure that matching details are included. This contribution is thus a case description method in support of future comparative research of immersive learning cases. We then discuss how the resulting description and interpretation can be leveraged to change immersion learning cases, by enriching them (considering low-effort changes or additions) or innovating (exploring more challenging avenues of transformation). The method holds significant promise to support better-grounded research in immersive learning.
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Recent theoretical progress indicates that spacetime and gravity emerge together from the entanglement structure of an underlying microscopic theory. These ideas are best understood in Anti-de Sitter space, where they rely on the area law for entanglement entropy. The extension to de Sitter space requires taking into account the entropy and temperature associated with the cosmological horizon. Using insights from string theory, black hole physics and quantum information theory we argue that the positive dark energy leads to a thermal volume law contribution to the entropy that overtakes the area law precisely at the cosmological horizon. Due to the competition between area and volume law entanglement the microscopic de Sitter states do not thermalise at sub-Hubble scales: they exhibit memory effects in the form of an entropy displacement caused by matter. The emergent laws of gravity contain an additional ‘dark’ gravitational force describing the ‘elastic’ response due to the entropy displacement. We derive an estimate of the strength of this extra force in terms of the baryonic mass, Newton’s constant and the Hubble acceleration scale a0 = cH0, and provide evidence for the fact that this additional ‘dark gravity force’ explains the observed phenomena in galaxies and clusters currently attributed to dark matter.
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The cost of information acquisition by natural selection
Ryan Seamus McGee, Olivia Kosterlitz, Artem Kaznatcheev, Benjamin Kerr, Carl T. Bergstrom
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karyotyping Practical Procedure
- 1. Karyotyping 1. Split cells that need to be karyotyped onto matrigel at 1/6 or 1/12 dilutions off from MEFs. 2. Cells should be in a stage of active division (not confluent) when initiating the karyotyping procedure. 3. Wear gloves. Add 23 ul of Vinblastine Sulfate (Vb) working solution (25ul/25ml ddH2O) to each well of a 6-well plate (23ul/3ml or 15ul/2ml Vb). Working conc of VB is 2 ug / ml. 4. Incubate cells 2 – 3 hours in 37o C incubator. 5. Meanwhile, put one bottle of sterile ddH20 and one 50ml tube filled with 70% Ethanol in fridge (4C). Also, put one box of slides at –20C. 6. 45 minutes prior to hood work, put hypotonic solution, 1x PBS, and wash media (DMEM + 5% FBS) in 37C water bath. 20 minutes prior to hood work, put trypsin in 37o C water bath. 7. After 2-3 hour incubation, prepare 3 15ml tubes with 8ml of wash media in each. Label as 15, 30, and 40. 8. Get cells from incubator and aspire media from wells, add 1ml 1x PBS/well, swirl and aspire. Repeat. 9. Add 1ml/well of warm trypsin and incubate for 1-2 min in 37C incubator. 10.Pipette cells up/down with P1000 and transfer 1/3 (2 wells) of cells to each 15ml tube. 11.Spin tubes 5 minutes @ 800rpm. 12.Remove sup by either aspiring it or pouring it out and resuspend pellet by flicking tube. 13.SLOWLY (i.e. drop by drop) add 2.3ml hypotonic solution per tube. Put tubes in 37C water bath for 15, 30, and 40 minutes. 14.When incubation period is over, take tube and spin 5 minutes @ 800rpm. 15.Meanwhile, make 50ml of Carnoyʼs fixative. It must be made FRESH 1:3 Glacial acetic acid: Methanol, 12.5ml Glacial acetic acid ,37.5ml Methanol
- 2. 14. Remove most of sup, and resuspend pellet in remaining media. 15.Using a dropper, slowly add 10 drops of Carnoyʼs fixative, flicking tube in between drops to prevent clumping. Bring volume to 2ml with fixative. 16.Spin 5 minutes. 17. Remove supernatant and slowly add 4ml of fixative, flick tube, spin and discard sup. Repeat, but donʼt remove all of sup. the second time around. Leave about 1ml of sup. and flick. 18.When you are ready to drop the cells, fill plastic box with ice and pour enough of the chilled ddH20 and Ethanol into two separate beakers to dip and cover the cold slides. Leave the beakers on ice. (Use slide holder for ethanol and large square beaker for ddH20). 19.Dip slides in chilled 70% ethanol and then rinse well with in the chilled ddH2O. Leave slides in the chilled water until you are ready to use them – slides must remain COLD in order for karyotype to work!!!. Using a glass pipette, aspire some of the cells and drop them onto the clean, wet and chilled slides either at arms length away or at varying heights from the floor. Dry slides on hot plate at LOW setting. 20.Stain with filtered Giemsa for 4 minutes. 21.Wash with 1x PBS for 5 minutes. 22.Wash with ddH2O. 23.Count at least 15 cells. Significant problem if more than 4 cells have more or less than 40 chromosomes (mouse) [or 46 for human]. Solutions for chromosome preparation Vinblastine sulfate salt. Sigma, cat# V1377 Add 0.5 ml sterile water to original vial containing 1mg of crystal vinblastine sulfate for a 2mg/ml stock solution. Aliquot and dilute to working solution of 25ul/25mlsterile water. Keep stock and working solution covered with foil at 4C. Hypotonic solution 1.5g sodium citrate 1.12 g potassium chloride q.s to .5L