How Silica Spin Column DNA and RNA Preps Work

We give a lot of troubleshooting help on DNA and RNA isolation here at Bitesize Bio because almost everything we do in molecular biology requires DNA or RNA at the very first step. These days, most labs use commercial kits, which employ spin columns, for the isolation of nucleic acids. The spin columns contain a silica resin that selectively binds DNA/RNA, depending on the salt conditions and other factors influenced by the extraction method.

These kits make the whole process much easier and faster than the methods of old, when things are going well, but the downside of using a kit is that we don’t always know what is in the mysterious and proprietary set of solutions that each company uses in its kit, which makes troubleshooting more difficult.

So in this article, I’ll explain in some detail how silica spin filter kits work and what is going on at each step. I’ll also go over some common problems specific to silica columns that can be overcome or avoided with just a little extra understanding.

Lysis:

The lysis formulas may vary based on the whether you want DNA or RNA, but the common denominator is a lysis buffer containing a high concentration of chaotropic salt. Chaotropes destabilize hydrogen bonds, van der Waals forces, and hydrophobic interactions. Proteins are destabilized, including nucleases, and the association of nucleic acids with water is disrupted setting up the conditions for the transfer to silica.

Chaotropic salts include guanidine HCL, guanidine thiocyanate, urea, and lithium perchlorate.

Besides the chaotropes, there is usually some detergents involved, to help with protein solubilization and lysis. There can also be enzymes used for lysis depending on the samples type. Proteinase K is one of these, and actually works very well in these denaturing buffers; the more denatured the protein, the better Proteinase K works. Lysozyme, however, does not work in the denaturing and so lysozyme treatment is usually done before adding the denaturing salts.

One comment about plasmid preps, the lysis is very different than extraction for RNA or genomic DNA because the plasmid has to be separated from the genomic DNA first and if you throw in chaotropes, you’ll release everything at once and won’t be able to differentially separate the small circular DNA from the high molecular weight chromosome. So, in plasmid preps the chaotropes are not added until after lysis and the salts are used for binding. An excellent in-depth article on alkaline lysis is here and also another article on the difference between genomic DNA and plasmid is available for further reading.

Binding:

The chaotropic salts are critical for lysis, but also for binding, as we discussed. Additionally, to enhance and influence the binding of nucleic acids to silica, alcohol is also added. Most of the time this is ethanol but sometimes it may be isopropanol. The percent ethanol and the volume has big effects. Too much and you’ll bring in a lot of degraded nucleic acids and small species that will influence UV260 readings and throw off some of your yields. Too little, and it may become difficult to wash away all of the salt from the membrane.

The important point here is that the ethanol influences binding and the amount added is optimized for whatever kit you are using. Modifying that step can help change what you recover so if you are having problems and want to troubleshoot recovery, that can be a step to evaluate further.

Another way to diagnose problems is to save the flow-through after binding and precipitate it to see if you can find the nucleic acids you are searching for. If you used an SDS-containing detergent in lysis, try using NaCl as a precipitant to avoid contamination of the DNA or RNA with detergent.

Washing Steps:

Your lysate was centrifuged through the silica membrane and now your DNA or RNA should be bound to the column and the impurities, protein and polysaccharides, should have passed through. But, the membrane is still dirty with residual proteins and salt. If the sample was from plants, there will still be polysaccharides, maybe some pigments too, left on the membrane, or if the sample was blood, the membrane might be tinted brown or yellow.

The wash steps serve to remove these impurities. There are typically two washes, although this can vary depending on the sample type. The first wash will often have a low amount of chaotropic salt to remove the protein and colored contaminants. This is always followed with an ethanol wash to remove the salts. If the prep is something that didn’t have a lot of protein to start, such as plasmid preps or PCR clean up, then only an ethanol wash is needed.

Removal of the chaotropic salts is crucial to getting high yields and purity DNA or RNA. Some kits will even wash the column with ethanol twice. If salt remains behind, the elution of nucleic acid is going to be poor, and the A230 reading will be high, resulting in low 260/230 ratios.

Dry Spin:

After the ethanol wash, most protocols have a centrifugation step to dry the column. This is to remove the ethanol and is essential for a clean eluant. When 10 mM Tris buffer or water is applied to the membrane for elution, the nucleic acids can become hydrated and will release from the membrane. If the column still has ethanol on it, then the nucleic acids cannot be fully rehydrated.

Skipping the drying step results in ethanol contamination and low yields. I do not see ethanol absorbance on the Nanodrop, so it won’t show up in your readings. The main indicators of a problem are that when you try to load the sample onto an agarose gel, the DNA will not sink. Even in the presence of loading dye. Another indicator is that if you put the sample in the -20C, it doesn’t freeze.

Elution:

The final step is the release of pure DNA or RNA from the silica. For DNA preps, 10 mM Tris at a pH between 8-9 is typically used. DNA is more stable at a slightly basic pH and will dissolve faster in a buffer. This is true even for DNA pellets. Water tends to have a low pH, as low as 4-5 and high molecular weight DNA may not completely rehydrate in the short time used for elution. Elution of DNA can be maximized by allowing the buffer to sit in the membrane for a few minutes before centrifugation.

RNA, on the other hand, is fine at a slightly acidic pH and so water is the preferred diluent. RNA dissolves readily in water.

What other things can go wrong:

Low yields: If you experience yields lower than you expected for a sample, there are many factors to think about. Usually it is a lysis problem. Incomplete lysis is a major cause of low yields. It could also be caused by incorrect binding conditions. Make sure to use fresh high quality ethanol (100% 200 proof) to dilute buffers or for adding to the binding step. Low quality ethanol or old stocks may have taken on water and not be the correct concentration. If the wash buffer is not made correctly, you may be washing off your DNA or RNA.

Low Purity: If the sample is contaminated with protein (low 260/280) then maybe you started with too much sample and the protein was not completely removed or dissolved. If the sample has poor 260/230 ratio the issue is usually salt from the bind or the wash buffer. Make sure that the highest quality ethanol was used to prepare wash buffers and if the problem continues, give the colun an additional wash.

Some samples have a lot more inhibitors compared to others. Environmental samples are especially prone to purity issues because humic substances are solubilized during extraction. Humics behave similarly to DNA and are difficult to remove from the silica column. For this type of sample, specialized techniques exist to remove the protein and humics prior to the column step.

Degradation: This is more of a concern for RNA preps and an article that gives specific advice is here. Mainly with RNA, degradation occurs from inproper storage of the sample or an inefficient lysis, assuming of course that you eluted with RNase-free water. For DNA preps, degradation is not a huge problem because for PCR, the DNA can be sheared and it works fine. But if you were hoping to not have so much sheared DNA, then you may have used too strong a lysis method.

PCR Clean-up Special Considerations: The easiest of all the techniques, because it is simply adding a high concentration of binding salts (typically between 3-5 volumes of salt per volumes of PCR reaction) and centrifugation through the column. So when PCR Clean-up kits fail, it can be particularly frustrating. The first question I ask people is “did you check the results of the PCR on a gel?” because you cannot UV check a PCR reaction and have an accurate reading. There is way too much in a PCR reaction absorbing UV at 260: nucleotides, detergents, salts, and primers. In my experience, a failure of a PCR clean-up kit to work frequently is caused by a PCR reaction that has failed and so there was nothing to clean up. But if you know you had a strong PCR product, the best approach is to just save your flow-through fraction after binding. If the DNA doesn’t bind, that’s where it is. You can always rescue it and then clean it up again. And then call tech support and ask for a replacement kit.

co-extract DNA and RNA

I am trying to co-extract DNA and RNA from body fluid stains (e.g. blood, semen and saliva) for forensic casework which may have very small amount of the biological sample. I am thinking of Allprep DNA/RNA micro kit from Qiagen. However, I was told that the RLT buffer was not designed for very small sample volumes or sizes so that the yield would be very low. It was recommended to use QIAamp DNA Investigator instead, but the DNA and RNA will be in the same tube which is not ideal for my experiment. If I want to separate DNA and RNA, I would like to use ATL and AL buffer from QIAamp DNA Investigator to start, and then transfer the lysate to AllPrep DNA spin column to separate the DNA and RNA. However, from my understanding, DNA binds to silica column at higher pH (8-9) but RNA binds at acidic pH (4-5). I was not sure the pH of the ATL and AL buffer from QIAamp DNA Investigator. I would like to have your advise.

Tagetes erectus, Marigold, Thin layer chromatography, Potential

Herbal medicines originated from the ancient use of wild plants. Today, with the possibility of carefully controlling the cultivation of medicinal plants and even improving them genetically, it is possible to develop and market a wide variety and quality of herbal drugs of consistent chemical composition and excellent quality to treat various diseases. One of the important constituent of Tagetes erectus may possess that quality.1 Tagetes erectus belong to the family Compositae. It is a small shrub; the plant grows upto a height of 1-3 feet and spreads about 0.5-1 feet. The leaf is arranged in opposite to subopposite with pinnately compound leaf. Margin is denate with oblong in shape bears green in colour with a blade length less than 2 inch. The leaves are 4 to 11cm long and very deeply pinnatifid, with the lobes lanceolate, coarsely and sharply toothed, and 1 to 2.5cm long , the flowers are pale to deep yellow.2

The leaves of Tagetes erectus were collected from the wild sources of Shirpur forest, and it was identified and authenticated by Dr. Sagar Kshirsagar, Dept. of Botany, SSVPS, College of science, Dhulia. A voucher specimen is placed in the Dept. of Pharmacognosy for further reference. The collected plants were washed, dried and were pulverized with the help of mechanical grinder and was passed through sieve no 40, and stored in closed vessels for future use. The fresh leaves were used for Microscopy Identification.

Garlic as nutraceutical

It is evident that garlic has been used as a food product across the globe. Today it became inseparable part of our diet. Scholars around the world have proved garlic for its medicinal use in treatment & prevention of certain diseases. It possesses both curative and preventative properties; new focus is on its use in prevention of heart attack and cancer. By studying these properties of garlic it can be categorised as nutraceuticals.

Benzopyrone, antitubercular, CHN analysis.

A number of natural and synthetic benzopyrone derivatives have been reported to exert anti microbial, anti tubercular and anti diabetic activity.1 Benzopyrones having chromen (γ- benzopyrone) moiety are associated with interesting physiological activities such as anti microbial, anti tubercular, antidiaetic, antiviral, anticancer, anti-inflammatory 2 etc.In view of these observations and our interest in the synthesis of biologically active biheterocycles possessing benzopyrone nucleus we modified benzopyrone ring to explore activities associated with this nucleus & evaluated them for anti tubercular activity.

suitably designed Informed Consent Form (ICF)

The study was approved by the Institutional ethics committee at kasturba hospital, manipal. A suitably designed Informed Consent Form (ICF) in different languages namely English, Kannada and Malayalam was prepared and used for the purpose of the study. In order to record the data for the study, a separate Case Record Form (CRF) was designed and used, which contains the details of the patients demographics, medical history, medication history, final diagnosis, laboratory investigations, study specific investigations, patient outcome analysis chart, adverse event reporting card, discharge medication and details of follow up visits. The American Urological Association Symptom Score3 (AUASS) was used to assess the severity of urinary symptoms and to check the effectiveness of the treatment. The AUA symptom index questionnaire consists of 7 questions. For the purpose of this study questions 2, 4 and 7 were assigned to irritative symptoms, while questions 1, 3, 5 and 6 were assigned to obstructive symptom sub scores. Total scores were classified as mild 0 to 7, moderate 8 to 19 and severe 20 to 35 symptoms, as recommended by the AUA measurement committee3. This questionnaire has been adopted worldwide and it is also known as the International Prostate Symptom Score (IPSS). The quality of life questionnaires4 were classified as delighted with score (0), pleased with score (1), mostly satisfied with score (2), mixed with score (3), mostly dissatisfied with score (4), unhappy with score (5) and terrible with score (6) was used to assess the quality of life of the patient with bladder outlet obstruction.This was a randomised controlled study conducted in the Dept. of Urology of Kasturba Hospital, Manipal from September 2008 to April 2009. Our first step was to identify the female patients with bladder outlet obstruction and male patients with BPH during ward rounds or with the help of the physician. Once the patient was identified, they were enrolled according to the study criteria and subject information sheet was explained in detail to the patient or patient‟s legally acceptable representatives and written inform consent was obtained from qualified patients prior to their enrolment. A detailed history, clinical examination, study specific investigations which includes Maximum Flow Rate (MFR), Post Voidal Residual Volume (PVR) and bladder thickness was done and the follow up details for all the patients was recorded in the CRF followed by assessment of American Urological Association symptom score (AUASS). The treatment patterns were analyzed with respect to medical treatment or surgical procedures adapted to the patient.

earthworms Eudrilus

The anthelminitic activity was carried out against earthworms Eudrilus species by Garg and Atal method at 2 mg/ ml concentration. Suspension of samples was prepared by triturating synthesized cyclic peptide (200 mg) with Tween 80 (0.5 %) and distilled water. The resulting mixture was stirred using mechanical stirrer for 30 min. The suspensions were diluted to contain 0.4 % w/v of the test samples. Suspension of the standard drug albendazole was prepared with the same concentration in a similar way.
Three sets of five earthworms of almost similar sizes were placed in petri plates containing 50 ml suspension of Tween 80 (0.5 %) and distilled water. The paralyzing and death times were noted and their mean was calculated for triplicate sets. The death time was ascertained by placing the earthworms in warm water (50ºC) which stimulated the movement.

Candida albicans and Asperigillus niger

The synthesized cyclic peptide was screened for antifungal activity against Candida albicans and Asperigillus niger. A spore suspension in normal saline was prepared from culture of the test fungi on sabouraud‟s broth media. After transferring growth media, petri plates were inoculated with spore suspension. After drying, wells were made using agar punch and the test samples, reference drug (griseofulvin) (50μg/ml) and negative control (DMSO) were placed in labeled wells in each petri plate. The test samples were tested at the concentrations 25, 50, 100 μg/ml. The petri plates inoculated with fungal cultures were incubated at 25ºC for 48 hrs. Diameters of the zone of inhibition were calculated in triplicate sets. The diameters obtained for the test sample were compared with that produced by the standard drug griseofulvin.

Antibacterial Activity

The synthesized cyclic peptide was screened for antibacterial activity against Escherichia coli, Staphylococcus aureus and Bacillus subtilis using modified Kirby-Bauer disc diffusion method. A spore suspension in sterile distilled water was prepared from five days old culture of the test bacteria growing on nutrient broth media. About 20 ml of the growth was transferred into sterilized petri plates and inoculated with 1.5 ml of the spore suspension. Each petri plate was divided into five equal portions along the diameter to place one disc. Three discs of test sample were placed on three portions together with one disc reference drug ciprofloxacin (50μg/ml) and a disc impregnated with the solvent DMF as negative control. The test samples were tested at the concentrations 25, 50, 100 μg/ml. The petri plates inoculated with bacterial cultures were incubated at 37ºC for 18 hrs. Diameters of the zone of inhibition were calculated in triplicate sets.

Synthesis of cyclic heptapeptide, cyclo(Gly-Tyr-Val-Pro-Leu-Trp-Pro)

To synthesize cyclo(Gly-Tyr-Val-Pro-Leu-Trp-Pro) (14), 9.44 gm of linear heptapeptide unit(10 mmol) (13) was deprotected at carboxyl end using 0.36 gm of LiOH (15 mmol) to get Boc-Tyr-Val-Pro-Leu-Trp-Pro-Gly-OH (13a) following the same procedure as adopted for the synthesis of compounds 8a and 9a from compounds 8 and 9 respectively.

The deprotected heptapeptide unit 13a (4.65 gm, 5 mmol) was dissolved in 50 ml of CHCl3 at 0ºC. To the above solution, 0.94 gm of pnp (6.7 mmol) was added and stirred at room temperature for 12 hrs. The reaction mixture was filtered and the filtrate was washed with 10% NaHCO3 solution (3 x 15 ml) until excess of pnp was removed and finally washed with 5% HCl (2 x 10 ml) to get the corresponding p-nitrophenyl ester Boc-Tyr-Val-Pro-Leu-Trp-Pro-Gly-O-pnp (13b). To compound 13b (4.20 gm, 4 mmol) dissolved in 35 ml of CHCl3, 0.91 gm of TFA (8 mmol) was added, stirred at room temperature for 1 hr and washed with 10% NaHCO3 solution (2 x 25 ml).

Tyr-Val-Pro-Leu-Trp-Pro-Gly-O-pnp (13c), which was dissolved in 25 ml of CHCl3 and 2.3 ml of NMM (21 mmol) was added. Then all the contents were kept at 0ºC for 7 days. The reaction mixture was washed with 10% NaHCO3 until the byproduct p-nitrophenol was removed completely and finally washed with 5% HCl (3 x 15 ml). The organic layer was dried over anhydrous sodium sulphate. Finally, chloroform was distilled off and the crude cyclized product was crystallized from CHCl3 and n-hexane to get the pure compound 14.

Synthesis of Boc-tri/tetrapeptide methyl esters

To synthesize Boc-Tyr-Val-Pro-OMe (11), 3.80 gm of dipeptide unit 8a (10 mmol) was coupled with 1.66 gm of amino acid methyl ester hydrochloride 4 (10 mmol) in the presence of DCC and NMM following the same procedure as adopted for the synthesis of Boc-dipeptide methyl esters 8-10. Similarly Boc-Leu-Trp-Pro-Gly-OMe (12) was prepared by coupling 3.36 gm of deprotected dipeptide unit 9a and 1.86 gm of 10a using DCC as the coupling agent and NMM as the base.

To synthesize Boc-Tyr-Val-Pro-Leu-Trp-Pro-Gly-OMe (13), 4.77 gm of tripeptide unit 11 mmol was deprotected at carboxyl end to get Boc-Tyr-Val-Pro-OH (11a) following the same procedure as adopted for the synthesis of compounds 8a and 9a from compounds 9 and 10, respectively. Tetrapeptide unit (12) (5.85 gm, 10 mmol) was deprotected at amino end to get Leu-Trp-Pro-Gly-OMe

Synthesis of L-amino acid methyl ester hydrochlorides

Thionyl chloride (0.73mL, 10 mmol) was slowly added to methanol (50 mL) at 0C and 1.15gm of L- proline (10 mmol) was added to the above solution. The resulting mixture was refluxed for 9 hrs at 110 ºC. Methanol was evaporated and the residue was triturated with ether at 0C until excess dimethyl sulphite was removed. The crude product was purified by recrystallization from methanol and ether at 0C to get L-proline methyl ester hydrochloride (4). Similarly, L-valine methyl ester hydrochloride (5), L-tryptophan methyl ester hydrochloride (6) and glycine methyl ester hydrochloride (7) were prepared by refluxing 1.17 gm of L-valine (10 mmol), 2.04 gm of L-tryptophan (10 mmol) and 0.75 gm of glycine (10 mmol) with 50 ml methanol in the presence of 0.73 ml of thionyl chloride (10 mmol).

Bioactive cyclic heptapeptide cyclo

A new bioactive cyclic heptapeptide cyclo(Gly-Tyr-Val-Pro-Leu-Trp-Pro) was synthesized using the solution phase technique by cyclization of the linear peptide Boc- Gly-Tyr-Val-Pro-Leu-Trp-Pro after proper deprotection at carboxyl and amino terminals. All the coupling reactions were performed at room temperature utilizing dicyclohexylcarbodiimide (DCC) as the coupling agent and N-methylmorpholine (NMM) as the base. Structures of all new compounds were characterized by IR and 1HNMR. The synthesized cyclopeptide was screened for antimicrobial and anthelminitic activities and found to exhibit good antibacterial activity against Bacillus subtilis and moderate antifungal activity against Candida albicans and Asperigillus niger. In addition the cyclic peptide was found to exhibit good anthelminitic activity against earthworms Eudrilus species.

Cyclic congeners possess unusual or modified amino acid residues and exhibit there bioactivities through binding to corresponding enzyme. This characteristic feature can allow bioactive cyclopeptides to act as therapeutic agents in this resistant world. Cyclopepetides having multiple peptide bonds are concerned with a wide spectrum of biological activities such as antimicrobial, anti-inflammatory, antimalarial, cyctotoxic, and antifungal activities. Cyclic peptides are more important compounds for medicinal purposes and represent an important class of natural products. Since only minute quantities are obtained from natural resources many of these compounds were attempted to synthesize in the laboratory. Keeping in view the biological potential of cyclic peptide as well as to obtain a bioactive compound in a good yield, the present investigation aimed at synthesis of cyclic heptapeptide cyclo(Gly-Tyr-Val-Pro-Leu-Trp-Pro) in a convenient and economical manner. Synthesized cyclic heptapeptide was evaluated for pharmacological activities. The antibacterial and antifungal activities were carried out against variety of pathogenic microorganism like Escherichia coli, Staphylococcus aureus, Bacillus subtilis and Candida albicans, Asperigillus niger. The anthelminitic activity was carried out against Eudrilus species of earthworms.

Synthesis of free acid

Sodium hydroxide (0.89gm, 22.4mmol) in 25 ml water was slowly added with stirring to 2.01gm of 5-bromo-2-hydroxyaldehyde (10mmol) and 0.94gm of chloroacetic acid (10mmol). The mixture was heated on heating mantle to remove all the liquid and the residue was treated with 30 ml water. The mixture was cooled and filtered and clear solution was acidified with dilute hydrochloric acid. The aqueous layer was extracted with diethyl ether (2 x 25 ml). Combined organic extracts were dried over anhydrous sodium sulphate.

Deprotection of dipeptide

Deprotection of dipeptide at amino end (7a): Compound 7 (3.42 gm, 10mmol) was dissolved in 15 ml of chloroform and treated with 2.28 gm of trifluoroacetic acid (20 mmol). The resulting solution was stirred at room temperature for 1 hr and washed with 25 ml of saturated sodium hydrogen carbonate solution. The organic layer was dried over anhydrous sodium sulphate and concentrated under reduced pressure. The crude product was purified by recrystallization from mixture of chloroform and light petroleum ether (b.p. 40-60C) to get pure Leu-Pro-OMe.

Bromosalicyaldehyde

Melting points were determined and uncorrected. The amino acids, di-tert-butyl pyrocarbonate (Boc2O), 5- Bromosalicyaldehyde, DCC and NMM were obtained from Spectrochem Limited, Himedia laboratories Limited mumbai and Sd-fine-chem Limited, Mumbai, India. The IR spectra were recorded on a Perkin Elmer Fourier transform infrared spectrophotometer using KBr pellets. The 1HNMR spectra were recorded on the Bruker Avance II-400 NMR spectrometer using CDCl3 as the solvent. The purity of all the compounds was controlled by TLC on silica gel G plates. Chloroform:Methanol (9:1 v/v) was used as developing solvent system and dark brown spots were detected on exposure to iodine vapours in a tightly closed chamber.

Anthelmintic Activity

The anthelminitic activity was carried out against earthworms Eudrilus species by Garg and Atal method at 2 mg/ ml concentration. Suspension of samples was prepared by triturating synthesized cyclic peptide (200 mg) with Tween 80 (0.5 %) and distilled water. Suspension of the standard drug albendazole was prepared with the same concentration in a similar way. The paralyzing and death times were noted and their mean was calculated for triplicate sets. The death time was ascertained by placing the earthworms in warm water (50ºC) which stimulated the movement.

Antibacterial Activity

The synthesized peptide derivatives were screened for antibacterial activity against Escherichia coli, Staphylococcus aureus and Bacillus subtilis using modified Kirby-Bauer disc diffusion method (DMF as a solvent).The test samples were tested at the concentrations 25, 50, 100 μg/ml. The petri plates inoculated with bacterial cultures were incubated at 37ºC for 18 hrs. The diameters obtained for the test sample were compared with that produced by the standard drug ciprofloxacin.

The synthesized peptide derivatives were screened for antifungal activity against Candida albicans and Asperigillus niger. DMSO is used as negative control. The test samples were tested at the concentrations 25, 50, 100 μg/ml. The petri plates inoculated with fungal cultures were incubated at 25ºC for 48 hrs. Diameters of the zone of inhibition were calculated in triplicate sets. The diameters obtained for the test sample were compared with that produced by the standard drug griseofulvin.

Phenoxyacetic acid, amino acids, antibacterial, antifungal and anthelminitic.

Phenoxyacetic acid is among the most vital moieties which are associate with potent antidiabetic (Rival et al 2004), antimycobacterial (Yar et al 2007),diuretic (Lebedev et al 1985, Woltersdorf et al 1976, Bicking et al 1976), anti-inflammatory (Kunsch et al 2005, Shokol et al 2005), antibiotic (Grardin et al 1995), anti-obesity (Kiso et al 1999), diagnostic (Ohmomo et al 1989), inhibition of platelet aggregation (Meanwell et al 1993, Seiler et al 1994) activities. The review of literature has suggested that incorporation of amino acids and peptides into aromatic and heterocyclic congeners have resulted in compounds with potent bioactivities.

Introducing an amino acid or peptide into aromatic compounds can increase the potency, decrease the toxicity and prolong its action. Among aromatics, phenolic compounds have wide range of activities. Further phenoxylation the resulting compound phenoxyacetic acid is obtained, which is well known for their biological potential. Thus keeping in view the biological potency of phenoxyacetic acids as well as taking advantage of biodegradability and biocompatibility of a novel series of substituted phenoxyacetic acid derivatives of amino acids and peptides have been synthesized with an anticipation to get potent agents with

Animal experiments

Pharmaceutical Undergraduate curriculum in India continues to follow an archaic syllabus set up decades ago, which has not kept pace with the technological progress. The Animal Physiology syllabus does not reflect the constraints imposed by the Prevention of Cruelty to Animals act of 1960 in the transport, care and experimentation.
The CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals) was established under Prevention of Cruelty to Animals act of 1960. The Experiments on Animals (Control and Supervision) Amendment Rules (1998) and the Breeding of and Experiments on Animals (Control and Supervision) Rules (1998) gave powers to the CPCSEA.