Isolation of Casein from Milk

  Isolation and Identification of Casein From Milk Course Notes

Milk is the probably the most nutritionally complete food found in nature. Whole milk contains vitamins (principally thiamine,riboflavin, panthothenic acid and vitamins A, B₁₂ and D), minerals (calcium, sodium, phosphorus, potassium, and trace minerals), proteins (which include all the essential amino acids), carbohydrates (mostly lactose), and lipids (fats). Whole milk is an oil in water emulsion, containing approximately 4% fat dispersed as very small (micron sized) globules. The fat emulsion is stabilized by complex phospholipids and proteins that are absorbed on the surface of the emulsion. Since the fat in milk is so finely dispersed it is more easily digested than fats from any other source.

Read Full Protocol Here

Isolation of Casein, Lactose, and Albumin from Milk

Milk is a food of exceptional interest. Not only is milk an excellent food for the very young, but humans 

have also adapted milk, specifically cow’s milk, as a food substance for persons of all ages. Many 

specialized milk products like cheese, yogurt, butter, and ice cream are staples of our diet.

Read Full Protocol Here.

Experiment 11: Isolation and Characterization of Casein from Milk

Adapted from Experiment 21, “Isolation of Protein, Carbohydrate and Fat from Milk”, in

Mohr. S.C., Griffin, S.F., and Gensler, W. J. Laboratory Manual for Fundamentals of

Organic and Biological Chemistry by John McMurry and Mary E. Castellion,: nglewood

Cliffs, Prentice-Hall, 1994 and Wayne P. Anderson (4/2002)

Read Full Experimental Protocol Here

ISOLATION OF CASEIN FROM MILK

Purpose: In this lab you will be isolating the proteins casein and lactalbumin from a sample of milk. You

will use these values to determine the percent protein in milk compared to the listed value on the box.

Read Full Experimental Protocol here.

 

Polony PCR Amplification

This section outlines the protocol for single-molecule amplification within the acrylamide gel.  The protocol given here may differ in specifics from what is presented here, but reflects what we are doing currently (Summer 2003).  We have tried to include excessive detail here but let us know if anything is unclear.  The general steps are:

1.    Cast acrylamide gels
2.    Diffuse in PCR reagents
3.    PCR amplification
4.    Slide clean-up


Further Protocol 

Long PCR Protocol Reagents and Guidelines

General Guidelines for Long PCR Conditions and Enzyme Mixtures
Efficient Long PCR results from the use of two polymerases: a non-proofreading polymerase is the main polymerase in the reaction, and a proofreading polymerase (3' to 5' exo) is present at a lower concentration. Following the results of Cheng et al. (1) we have had success using Tth (ABI/Perkin-Elmer) as the main-component polymerase and Vent (New England Biolabs) as the fractional-component polymerase. Other combinations of proofreading and non-proofreading polymerases have been used successfully for many applications. The buffer listed below works well with Tth and Vent, but not with others. If you are interested in using other polymerases make sure that you use compatible buffers. Error rates for other polymerases can be found at http://research.nwfsc.noaa.gov/protocols/taq-errors.html 

Read Full Protocol Here

Qiagen RNeasy Plant RNA Isolation

RNA isolation

RNeasy Kits are designed to isolate total RNA from small quantities of starting material. They provide a fast and simple method for the preparation of up to 100 µg total RNA from animal cells and tissues, bacteria, and yeast (RNeasy Mini Kits) or plant cells and tissues and filamentous fungi (RNeasy Plant Mini Kits). RNeasy Plant Mini Kits are fast and avoid tedious methods, such as CsCl step-gradient ultracentrifugation and alcohol precipitation steps, or methods involving the use of toxic substances such as phenol and/or chloroform. The purified RNA is ready for use in standard downstream applications such as RT-PCR, Northern, dot, and slot blotting, poly A+ RNA selection, primer extension, RNase and S1 nuclease protection, cDNA synthesis, differential display, expression-array and expression-chip analysis.

Further Protocol Information

Formaldehyde Agarose Gel Electrophoresis Protocol

The following protocol for formaldehyde-agarose gel electrophoresis gives enhanced sensitivity for gel and subsequent analysis (e.g. northern blotting). A key feature is the concentrated RNA loading buffer that allows a larger volume of RNA sample to be loaded onto the gel than conventional protocols (e.g. Sambrook, J. et al., eds. (1989) Molecular cloning — a laboratory manual, 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press).

1.2% Formaldehyde Agarose gel preparation

To prepare Formaldehyde Agarose gel (1.2% agarose) of size 10 x 14 x 0.7 cm, mix:

1.2 g agarose

10 ml 10x Formaldehyde Agarose gel buffer (see composition below)

Add RNase-free water to 100 ml

If smaller or larger gels are needed, adjust the quantities of components proportionately. Heat the mixture to melt agarose. Cool to 65°C in a water bath. Add 1.8 ml of 37% (12.3 M) formaldehyde (toxic) and 1 µl of a 10 mg/ml ethidium bromide (mutagenic) stock solution. Mix thoroughly and pour onto gel support. Prior to running the gel, equilibrate in 1x Formaldehyde Agarose gel running buffer for at least 30 min.

RNA sample preparation for Formaldehyde Agarose gel electrophoresis

Add 1 volume of 5x loading buffer per 4 volumes of RNA sample (for example 10 µl of loading buffer and 40 µl of RNA) and mix.

Incubate for 3–5 min at 65°C, chill on ice, and load onto the equilibrated Formaldehyde Agarose gel.

Gel running conditions

Run gel at 5–7 V/cm in 1x Formaldehyde Agarose gel running buffer.

Composition of Formaldehyde Agarose gel buffers

10x Formaldehyde Agarose Gel buffer

200 mM 3-[N-morpholino]propanesulfonic acid (MOPS) (free acid)

50 mM sodium acetate

10 mM EDTA

pH to 7.0 with NaOH

1x Formaldehyde Agarose Gel Running Buffer

100 ml 10x Formaldehyde Agarose gel buffer

20 ml 37% (12.3 M) formaldehyde

880 ml RNase-free water

5x RNA Loading Buffer

16 µl saturated aqueous bromophenol blue solution†

80 µl 500 mM EDTA, pH 8.0

720 µl 37% (12.3 M) formaldehyde

2 ml 100% glycerol

3084 µl formamide

4 ml 10 x Formaldehyde Agarose gel buffer

RNase-free water to 10 ml

Stability: Approximately 3 months at 4°C

Ref

2M H2SO4 for 100ml

2M H₂SO₄ for 100ml

• mix 50ml Milli Q water + 50ml 4M H₂SO₄ solution

4M H2SO4 for 1 liter

4M H₂SO₄ for 1 liter

• Carefully pour 392ml H₂SO₄ {Sulphuric acid 96% H₂SO₄ Carlo Erba reagents Code no. 410301 (Reanal 17769) FW 98.078} into 608ml Milli Q water (always pour acide into water)

TMB-buffer for 1 liter

TMB-buffer for 1 liter[0.1M Na-acetate, pH 5.5]

• Disolve in 950ml Milli Q water
• 13.6g Na-acetate {Nátrium acetát, kristályvizes CH₃COONa*3H₂O M:136.08 Reanal 14021}
• Adjust pH to 5.5 with app. 700ul cc. Acetic acid {Ecetsav 96% Reanal 0914-1-08-65 M:60.05}
• Adjust volume to 1 liter with Milli Q water 

TMB solution for 1ml - ELISA

TMB solution for 1ml [10mg/ml TNB in DMSO]

• Disolve in 1ml DMSO {Dimethyl sulfoxide, minimum 99.5% GC Sigma D5879-100ml }
• 10mg TNP {3,3',5,5'-tetramethyl-benzidine Sigma T-2885 FW240.3}
• Store at +4C

ELISA blocking-buffer for 10ml

ELISA blocking-buffer for 10ml [0.05% Tween 20, 5%BSA, 0.05% azide, PBS]

• Mix on magnetic stirrer:
• 10ml PBS-Tween
• 100mg BSA {Sigma, No.:A3059-100G, Albumin, bovine serum, Fraction V}
• 50ul Sodium azide (from 10% NaN₃ stock solution)
• Store at +4C

Carbonate buffer pH 9.5 for 1 liter (for ELISA coat)

Carbonate buffer pH 9.5 for 1 liter (for ELISA coat) Disolve in 1 liter Milli Q water: 1.6g Na2CO3 2.9g NaHCO3 Adjust to pH 9.5 Store at R/T

ELISA Method

Method Coat ELISA plate with 50ul/well 0.2-10ug/ml protein (purity should be above 3%) in PBS or carbonate buffer for 2h on R/T or O/N in the fridge Wash 3x with PBS-Tween (alternative: following washing incubate in ELISA-blocking buffer for 30min R/T and wash 3x with PBS-Tween) Incubate in PBS-Tween diluted antibody/protein solution for 1h at 37C (ideally after 5h reach the equibrium) Wash 3x with PBS-Tween Develope with TMB Add 100ul TMB developing solution to wells For 1 ELISA plate mix: 11ml TMB buffer 110ul TMB solution 22ul H2O2 {Hydrogen peroxide 30% solution Sigma H-1009} Add 100ul 2M H2SO4 to stop the reaction Detect absorbance at 450nm

5x Veronal buffer for 500ml (5xVBS) Recipe

5x Veronal buffer for 500ml (5xVBS) [727mM NaCl, 9.12mM Na-diethyl-barbiturate (C8H11O3N2Na), 15.63mM 5,5’-diethyl barbiture acid (C8H12N2O3), pH7.3] Make solution 'A': 0.94g Na-diethyl-barbiturate (C8H11O3N2Na){5,5-Diathylbarbitursaure. Na-salz Serva 18797; MW:206.2} 21.25 g NaCl {Reanal, No.:2464-1-22-38, Nátrium-klorid, Ph. Eur.5; MW:58.44} ~300ml Milli Q water Make solution 'B': 1.44g 5,5’- diethyl barbiture acid {5,5-dietil-barbitursav Reanal 04049; MW:184.2} ~100ml Milli Q water Boil it otherwise it won’t dissolve, then cool it down Mix solution ‘A’ and ‘B’ set pH to 7.3 with app. 50-70μl 10N NaOH Fill up until 500ml with Milli Q water, then store it at +4C

1x Veronal buffer recipe

1x Veronal buffer (VBS)[145mM NaCl, 1,8mM Na-diethyl-barbiturate (C8H11O3N2Na), 3mM 5,5’-diethyl barbiture acid (C8H12N2O3), pH7.3] Dilute 5x Veronal buffer with Milli Q water

0.2M EGTA stock solution for 10ml

0.2M EGTA stock solution for 10ml Mix on magnetic stirrer: 8ml Milli Q water 0.7608g EGTA{ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) Sigma E-4378 LOT 111K5411 FW 380,4} Adjust to pH8 with 10N NaOH otherwise it won't disolve (be careful EGTA is an acid) Adjust volume to 10ml with Milli Q water Store at 4C

EDTA-VBS solution for 10ml

EDTA-VBS solution for 10ml (dilute serum 5x with it)[25mM EDTA, 5%BSA, 0.05% Tween 20, VBS ] Mix on magnetic stirrer: 2ml 5x VBS 500ul 0.5M EDTA 0.5g BSA {Sigma, No.:A3059-100G, Albumin, bovine serum, Fraction V} 50ul 10% Tween 20 {Sigma No.:P-1379, Polyoxylethylene-sorbitan monolaurate (Tween 20)} Adjust volume to 10ml with Milli Q water Filtrate though an 0.45um filter Aliquot 1ml/eppendorf Store at -20C

Mg-EGTA-VBS solution for 10ml

Mg-EGTA-VBS solution for 10ml (dilute serum 5x with it)[2.5mM Mg2+, 6.2mM EGTA, 5%BSA, 0.05% Tween 20, VBS] Mix on magnetic stirrer: 2ml 5x VBS 310ul 0.2M EGTA 25ul 1M MgCl2 stock solution 0.5g BSA {Sigma, No.:A3059-100G, Albumin, bovine serum, Fraction V} 50ul 10% Tween 20 {Sigma No.:P-1379, Polyoxylethylene-sorbitan monolaurate (Tween 20)} Adjust volume to 10ml with Milli Q water Filtrate though an 0.45um filter Aliquot 1ml/eppendorf Store at -20C

Ca-Mg-VBS (veronal buffered saline) for 25ml

Ca-Mg-VBS (veronal buffered saline) for 25ml (for serum dilution)[2.5mM Ca2+, 0.7mM Mg2+, 0.05% Tween 20, 5%BSA, VBS] Mix on magnetic stirrer: 5ml 5x VBS 250ul Ca2+ Mg2+ stock solution [0.25M Ca2+, 0.07M Mg2+] 1.25g BSA {Sigma, No.:A3059-100G, Albumin, bovine serum, Fraction V} 12.5ul Tween 20 {Sigma No.:P-1379, Polyoxylethylene-sorbitan monolaurate (Tween 20)} Adjust volume to 25ml with Milli Q water Filtrate though an 0.45um filter Aliquot 1ml/eppendorf Store at -20C

Ca2+ Mg2+ stock solution for 5ml [0.25M Ca2+, 0.07M Mg2+]

Ca2+ Mg2+ stock solution for 5ml [0.25M Ca2+, 0.07M Mg2+] Dissolve in 5 ml Milli Q water: 0.1838g CaCl2*2H2O {Reanal 11024 MSZ 24161-65 Kalcium-klorid, szárított MW: 147.02 CaCl2*2H2O} 0.0712g MgCl2*6H2O {Reanal No.: 20281-1-01-38 Magnézium-klorid 6-hidrát MW: 203.30 MgCl2*6H2O}

PBS-Tween BSA for 300ml [0.05% Tween 20, 5%BSA, 0.05% azide, PBS]

PBS-Tween BSA for 300ml [0.05% Tween 20, 5%BSA, 0.05% azide, PBS] Mix on magnetic stirrer: 300ml PBS-Tween 15g BSA {Sigma, No.:A3059-100G, Albumin, bovine serum, Fraction V} 1.5ml Sodium azide (from 10% NaN3 stock solution) Filtrate through an 0.45um filter Aliquot 50ml/falcon Store at 4°C

PBS-Tween for 1 liter [0.05% Tween 20, PBS]

PBS-Tween for 1 liter [0.05% Tween 20, PBS] Mix on magnetic stirrer: 0.5ml Tween 20 {Sigma No.:P-1379, Polyoxylethylene-sorbitan monolaurate (Tween 20)} 1 liter PBS

1x PBS Recipe for 1 liter[137mM NaCl, 2.7mM KCl, 8mM Na2HPO4, 1.46mM KH2PO4]

Dissolve in 800 ml distilled water: 8 g NaCl {Reanal, No.:2464-1-22-38, Nátrium-klorid, Ph. Eur.5; MW:58.44} 0.2 g KCl {Reanal, No.:18050-1-01-38, Kálium-klorid, a.r.; MW: 74.56} 1.424 g Na2HPO4*2H2O {Reanal, No.:08973-1-01-38, di-Nátrium-hidrogén-foszfát 2-hidrát, a.r.; MW:177,99} 0.2 g KH2PO4 {Reanal, No.:17890-01-38, Kálium-dihidrogén-foszfát, a.r.; MW:136.09} Adjust volume to 1 liter with H2O Filtrate trough an 0.22um filter or sterilize by autoclave Store at room temperature

10x PBS Recipe for 1 liter

10x PBS Recipe for 1 liter Dissolve in 800 ml distilled water: 80 g NaCl {Reanal, No.:2464-1-22-38, Nátrium-klorid, Ph. Eur.5; MW:58.44} 2 g KCl {Reanal, No.:18050-1-01-38, Kálium-klorid, a.r.; MW: 74.56} 14.24 g Na2HPO4*2H2O {Reanal, No.:08973-1-01-38, di-Nátrium-hidrogén-foszfát 2-hidrát, a.r.; MW:177,99} 2 g KH2PO4 {Reanal, No.:17890-01-38, Kálium-dihidrogén-foszfát, a.r.; MW:136.09} Adjust volume to 1 liter with H2O Filtrate through an 0.22um filter and divide in 500ml aliquotes Store at room temperature

RNA Isolation Protocol from Tissue Samples

Place the desired tissue in the prechilled mortar, add liquid nitrogen and grind to a fine powder with the pestle. .. Transfer the ground tissue to a Falcon 2059 tube and add 1ml monophasic isolation reagent per 100mg tissue. .. Homogenize the tissue for 15-30 seconds with a polytron mechanical homogenizer. .. Incubate the homogenized sample for 5 minutes at RT to allow complete dissociation of protein complexes. Read Full protocol here.

Amyloid Beta HFIP Stock Protocol

Lyophilized peptide should be ideally ordered in 10 mg glass vials. In-house material should be accurately weighed in clean glass vials with a HFIP resistant closure. Read Full protocol Here.

Fluorimetric Enzyme Assay Protocol for Marine Samples

http://allison.bio.uci.edu/Protocols/EnzymeProtocolMarine.pdf

GRAM STAIN PROTOCOL - fecal smear to observe the bacterial microflora

This technique is used to stain a slide such as a fecal smear to observe the bacterial microflora present based on their gram stain reaction. “Heat-fix” the slide with the specimen by passing it over a heat source, such as a flame, several times using a forceps. The slide should be passed very quickly through the flame and not be heated excessively. Place slide on the staining tray. Read Full Protocol Here

HIV/AIDS Vaccine Developed in Canada

HIV/AIDS vaccine developed at Western proceeding to human clinical trials. After two decades of research, a group of Canadian scientists has won approval to start testing an experimental HIV vaccine on humans.

How Bubble Gum is Made?

Check this out. If you don't know how Gum is made.

How to make 1x Trypsin

Use the formula C1V1=C2V2 to make the dilution for your required volume.


C=Concentration
V=Volum

EXAMPLE
Make 20 ml of 1X Trypsin from a 10X stock.

        10X stock *  X  =  1X stock *  20 ml

                     X = (1X stock *  20 ml) / 10X stock

                     X = 2.0 ul 10X stock
                       +18.0 ul of PBS

How to make 1x TAE Buffer

To make 1X TAE take 20mL of 50X TAE and fill up the remaining volume of a
1000mL erlenmeyer flask with nano-pure water.

How to make 1x tae from 50x tae


Add 1ml of 50x to 49 ml of water. Increase the volume as required.

How to make 1x pbs from 10x pbs

If you have tablets then make the 10x pbs solution according the the manufacturer's instructions.

Than use the formula C1V1=C2V2 to make the dilution for your required volume.
C=Concentration
V=Volum


Make 20 ml of 1X PBS buffer from a 10X stock.

        10X stock *  X  =  1X stock *  20 ml

                     X = (1X stock *  20 ml) / 10X stock

                     X = 2.0 ul 10X stock
                       +18.0 ul of H2O

how to make 1x from 50x

You need to use the formula

C1V1=C2V2
C=Concentration
V=Volume

Example

Make 20 ul of 1X restriction enzyme buffer from a 10X stock.

        10X stock *  X  =  1X stock *  20 ul

                     X = (1X stock *  20 ul) / 10X stock

                     X = 2.0 ul 10X stock
                       +18.0 ul of H2O

How to make 1x from 10x

Simple Way to make Easy Dilutions - how to make 1x solution from 10x solution


10x means 10 Times concentrated. If you want to make 1x then you have to dilute it 10 time. For Example. To make 10 ml of 1x solution in water. You need take 1 ml of solution [10px] and mix with 9 ml of water. Final total volume is now 10 ml [10 times of 1ml] Done :)

Theoretical Way to make dilutions


You should use the formula C_i V_i = C_f V<sub>f 
or


M1V1=M2V2</sub>


Example 


Make 20 ul of 1X restriction enzyme buffer from a 10X stock.

        10X stock *  X  =  1X stock *  20 ul

                     X = (1X stock *  20 ul) / 10X stock

                     X = 2.0 ul 10X stock
                       +18.0 ul of H2O

 Amit

Isolation of Bacteria from the Environment - References

Isolation of bacteria from the environment http://delrio.dcccd.edu/jreynolds/microbiology/2420/files/bacteria_ubiquity.pdf

Laboratory Notes for BIO 1003 John H. Wahlert & Mary Jean Holland Examination of dental bacteria http://faculty.baruch.cuny.edu/jwahlert/bio1003/eubacteria.html

Culture and isolation of phototrophic sulfur bacteria from the marine environment http://adsabs.harvard.edu/abs/1970HWM....20....6T

Isolation of Bacteria from Soil Samples

ISOLATION OF SOIL BACTERIA: VIABLE TITER and PURE CULTURE
http://www2.fiu.edu/~makemson/MCB2000Lab/Exp4SoilBact.pdf

Isolation of an Unknown Bacterium from Soil
Department of Biological Sciences
University of Nevada, Las Vegas
http://www.ableweb.org/volumes/vol-14/6-steubing.pdf


ISOLATION OF PHOSPHATE SOLUBILISING BACTERIA FROM SOIL AND ITS ACTIVITY
http://biotechindia.files.wordpress.com/2007/12/isolation.pdf

Isolation of Soil Microorganisms
http://apps.caes.uga.edu/sbof/main/lessonPlan/microorganismIsolation.pdf


Isolation of bacteria from mechanic workshops’ soil environment contaminated with used engine oil
Department of Medical Laboratory Sciences, College of Medicine, University of Nigeria, Enugu Campus, Enugu, Nigeria.


Isolation of soil Streptomyces as source antibiotics active against antibiotic-resistant bacteria
http://ejobios.com/pdf/ejob-8-11-2,9,73-82.pdf


ISOLATION OF SOIL BACTERIA FOR BIOREMEDIATION OF HYDROCARBON CONTAMINATION
http://www.chem.msu.su/rus/vmgu/031/88.pdf

RNA isolation from soil for bacterial community and functional analysis: evaluation of different extraction and soil conservation protocols
http://www.engr.colostate.edu/~apruden/classes/ce742/Readings/Articles/RNAextractTechniques.pdf

TEM-PCR Method,Technology and Testing

What is TEM-PCR Technology?

The TEM-PCR stands for Target Enriched Multiplexing Polymerase Chain Reaction. TEM-PCR is a molecular multiplex PCR technology used Molecular Differential Detection. By TEM-PCR the molecular targets can be amplified in one reaction. This technology is advantageous over the RT-PCR Technology because multiple molecular targets can be amplified specifically in a single reaction. This makes it a important tool for detection of molecular markers of pathogens.

In last few years improvements have been done in this technology. Benson et. al. compared the ResPlex I assay kit of Qiagen with RT-PCR in the detection of various bacterial strains of respiratory disease and found that it is less sensitive than real-time PCR but has advantage over multiple assays [ref]. Deng et. al. detected more S. pneumoniae (32 vs. 7) and H. influenzae (29 vs. 23) by TEM-PCR than did culture [ref]. Related: Overlap Extension PCR

Isolation of Actinomycetes

References for Isolation of Actinomycetes

Culturable marine actinomycete diversity from tropical Pacific Ocean sediments.

Jensen PR, Gontang E, Mafnas C, Mincer TJ, Fenical W.

Source

Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California - San Diego, La Jolla, CA 92093-0204, USA
http://www.ncbi.nlm.nih.gov/pubmed/15946301

Isolation and characterization of a sponge-associated actinomycete that produces manzamines Marine-Estuarine-Environmental Sciences http://drum.lib.umd.edu/handle/1903/4114

Isolation of Acid Phosphatase

Here we have searched and compiled a list of methods used by various college and universities for the isolation of acid phosphatase.



Purification of Acid Phosphatase from Wheat Germ
There are three major steps involved in the purification of macromolecules.  First, you must disrupt the cell you are using as a source.  Second, you must selectively purify the macromolecule away from contaminating molecules.  Third, you must both prevent degradation of the macromolecule during the procedure and you must preserve the native structure of the macromolecule to be purified.  In the purification of proteins, you want to separate that protein from other “contaminating” proteins, and you want to purify it in its native conformation with retention of enzymatic activity.
Further Details

Purification and Partial Characterization of an Acid Phosphatase fromSpirodela oligorrhiza and Its Affinity for Selected Organophosphate Pesticides

Christopher F. Hoehamer,^†‡ Chris S. Mazur,*^‡ and N. L. Wolfe^‡

National Research Council, and National Exposure Research Laboratory, U.S. Environmental Protection Agency, 960 College Station Road, Athens, Georgia 30605http://pubs.acs.org/doi/abs/10.1021/jf040329u

Isolation and partial characterization of an acid phosphatase

from Artemisia vulgaris pollen extract

TANJA D. ]IRKOVI]*#, MARIJA DJ. GAVROVI]-JANKULOVI]* #, MIRJANA N. BUKILICA**,

LJUBA MANDI]*#, SPOMENKA Z. PETROVI]*** and RATKO M. JANKOV*

http://www.doiserbia.nb.rs/img/doi/0352-5139/2002/0352-51390209567C.pdf

Isolation of Acetylsalicylic Acid from Aspirin Tablets

Here we have compiled a list of laboratory protocols from various colleges and universities for the Isolation of acetylsalicylic acid from aspirin tablets.

Isolation of acetylsalicylic acid from aspirin tablets

Organic Chemistry Laboratory CHE 327 

Cornell College Term 7, 2004/2005

Addison Ault, Andrea Pionek, Mary Anne Teague, Charley Liberko

http://people.cornellcollege.edu/cliberko/OrgLabManual.htm


Organic Chemistry Laboratory
Isolation of Acetysalicylic Acid from Aspirin Tablets


Aspirin, like most tablets, contains the active ingredient (acetylsalicylic acid) plus a binder to give the tablet its shape.  The active ingredient in aspirin will be recovered by recrystallization from ethanol.  The aspirin tablets you will be using contain 325 mg of acetylsalicylic acid per tablet.  (However, check the bottle to be certain, as occasionally substitutions have to be made.)

From Mansfield.edu [PDF]


Synthesis of Acetylsalicylic Acid (Aspirin)


Introduction: The purpose of this experiment is to synthesize acetylsalicylic acid via an esterification
reaction between salicylic acid and acetic anhydride.  The product will be recrystallized using
95% ethanol. The product will then be analyzed via infrared spectroscopy, nuclear magnetic
resonance spectroscopy, ultraviolet light visible spectroscopy, and melting point analysis. It
will then be compared with the IR, NMR, and UV spectra and melting point of acetylsalicylic
acid isolated from commercial aspirin
http://www.franklincollege.edu/pwp/lmonroe/Organic%20Chem/Aspirin.pdf

Lysis buffer: RIPA or NP40 for Immunoprecipitation

Lysis buffer.

The choice of lysis buffer depends on what kind of IP you are doing.

RIPA buffer gives the lowest background, but can denature some kinases. It also has the potential to disrupt protein:protein interactions.

NP40 buffer (EVF) is less denaturing, but gives a higher background. It is less likely to inhibit kinase activity and disrupt protein complexes.

RIPA Buffer recipe

Further Buffer Information

RIPA Lysis Buffer Recipe : RIPA buffer for mammalian cell lysis:

RIPA buffer for mammalian cell lysis:
50mM Tris-cl pH 7.4
150mM NaCl
1% NP40
0.25% Na-deoxycholate
1mM PMSF
1x Roche complete mini protease inhibitor cocktail
1x Pierce phosphatase inhibitor cocktail

Stock solutions used:
100mM Tris-Cl pH 7.4, 300mM NaCl
10% NP40 in water
10% Na-deoxycholate in water (stored at RT in dark)
200mM PMSF in isopropanol
Roche complete protease inhibitor cocktail tablets (cat# 04 693 124 001)
Pierce phosphatase inhibitor cocktail (100x) (cat # 78420)
For 10ml of lysis buffer:
5ml  100mM Tris-Cl pH 7.4, 300mM NaCl
1ml  10% NP40 in water
3.65ml  water
250ul  10% Na-deoxycholate in water
1 tablet * Roche complete protease inhibitor cocktail
100ul*  Pierce phosphatase inhibitor cocktail
50ul*  200mM PMSF in isopropanol

*add at time of use. PMSF is only good for ~30minutes after dilution into buffer.

Reference [pdf]

RIPA Buffer Recipe

EDTA-free RIPA (RIPA[-]):
0.1% NP40, 20mM Tris/HCL pH
8,10% Glycerol, store at 4ºC (Usually
RIPA buffer contains 1 mM EDTA

Reference

10x PBS Buffer Recipe

10X PBS BUFFER
To make 1 Liter

1. Dissolve the following in ~800mL18 MΩ dH2O.  May need to heat a little to dissolve completely.
80g NaCl  14.4g Na2HPO4–7H2O (dibasic)
  2g KCl    2.4g KH2PO4 (monobasic)
2. Adjust pH to 7.4 with HCl or NaOH.
3. Raise volume to 1 liter.
4. Transfer to a 1L bottle, label and autoclave.

Reference

10X, 20X TRIS Buffer Recipes

10X Tris-HCl (0.5M Tris Base, pH7.6):
      Trizma Base ---------------------------------- 61 g
      Distilled water ------------------------------- 1000 ml
      Adjust pH 7.6 using concentrated HCl
       Store this solution at room temperature. Dilute 1:10 with distilled water before use
and adjust pH if necessary.
 
20X Tris-HCl (1M Tris Base, pH7.6):
      Trizma Base ---------------------------------- 122 g
      Distilled water ------------------------------- 1000 ml
      Adjust pH 7.6 using concentrated HCl
      Store this solution at room temperature. Dilute 1:20 with distilled water before use
and adjust pH if necessary.
 
10X Tris-HCl-Tween 20 (0.5M Tris Base, 0.5% Tween 20, pH7.6):
      Trizma Base ---------------------------------- 61 g
      Distilled water ------------------------------- 1000 ml
      Adjust pH 7.6 using concentrated HCl and then add 5 ml of Tween 20.
       Store this solution at room temperature. Dilute 1:10 with distilled water before use
and adjust pH if necessary.
 
Reference

10% SDS Recipe

10% SDS  Recipe

1L:  

100g SDS into 1 L, heat to 68C for solubility. pH ~6.6.

50X TAE DNA Electrophoresis Buffer Recipe

 TAE DNA Electrophoresis Buffer (50 X)

     (2 M Tris, 50 mM EDTA)    
     
     2 L
     484 g Tris
     114.2 ml glacial acetic acid
     200 ml 0.5 M EDTA 8.0  
To make 1x TAE 20 L, add 400 ml 50X buffer into 19.6 L ddH2O

Reference

0.5M EDTA pH 8.0 Recipe

EDTA 0.5 M (pH8.0)

    0.5M, 1L:  148 g EDTA
                       + ~30-40 g NaOH to adjust pH
                       (or 186 g EDTA-Na.2H2O + ~20 g NaOH)
   Note: pH adjusted by NaOH is essential for solubility. Autoclavable

Reference

1 M TRIS Buffer Recipe

1 M Tris-HCl Buffers
 
pH        Volume (L)              TrisBase (g)         HCl (ml)
pH 7.0  2                             242.2                  150-155
pH 7.5  2                             242.2                  120-125
pH 8.0  2                             242.2                   80-85

Autoclavable

Reference

0.5 x TBE Buffer Recipe

0.5 x tbe buffer recipe


-108 g Tris base
-55 g Boric acid
-40 ml 0.5 M EDTA pH 8.0
-H2O to 2 L

Place the flask on a magnetic stirrer until completely dissolved. This results in a 5X concentrated
stock of TBE Buffer. Dilute the 2L of 5X stock by 10 in distilled water (add 18 L or 4.5 gallons of
distilled water). Mix well. The buffer is ready for use.

Reference