Purification of Plasmid DNA

After the initial characterization, it is possible to purify further some or all of the plasmid DNAs by RNase digestion and extraction with organic solvents. This further purified DNA is suitable for techniques such as DNA sequencing, subcloning or the production of gene probes. In order to purify plasmid DNA after the isolation process, any residual RNA and contaminating protein are removed. This purification step involves two main steps, which are, first, removing residual RNA by using RNase in order to digest RNA and, second, extract contaminating protein using organic solvents, phenol-chloroform.

Estimation of Disulfide Bonds Using Ellman’s Reagent

Ellman’s reagent has been widely used for the quantitation of thiols in peptides and proteins. It has also been used to assay disulfides present after blocking any free thiols (e.g., by carboxymethylation) and reducing the disulfides prior to reaction with the reagent. It is also commonly used to check the efficiency of conjugation of sulfhydryl- containing peptides to carrier proteins in the production of antibodies.

Separation of DNA Fragments Using PAGE Method

This method is able to separate DNA fragments with the size of as small as 10 bp and up to 1 kb with the resolution of as little as 1 bp. While agarose gel electrophoresis is only able to separate DNA fragments with the bigger size that PAGE does or in the size range of 100 nucleotides to around 10 – 15 kb.

Polyacrylamide Gel Electrophoresis: Advantages and Disadvantages

You must have known that agarose gel electrophoresis is generally adequate for resolving nucleic acid fragments in the size range of 100 nucleotides to around 10-15 kb. But, for nucleic acid which its fragments below those range, it will be difficult to separate and hard to visualize because of diffusion within the gel matrix. These problems are solved by native polyacrylamide gel electrophoresis (PAGE). Using native PAGE, fragments as small as 10 bp and up to 1 kb can be separated with a resolution of as little as 1 bp.

Rapid Boiling Method for Plasmid DNA Isolation

In the previous post, it had already explained about how to extract plasmid DNA by using alkaline lysis method. In this particular post, it will be explained the other method to extract plasmid DNA instead of using alkaline lysis method. It is Rapid Boiling method, an alternative to alkaline lysis method that was developed by Holmes and Quigley. Here, the cells are lysed partially allowing plasmids to escape, whereas the bacterial chromosomal DNA remains trapped in the cell debris. High temperature is then used to denature the chromosomal DNA, after which reannealing allows the plasmids to reassociate. Centrifugation removes the chromosomal DNA along with the cell debris, leaving the plasmid in suspension, from where it is recovered by isopropanol precipitation.

Plasmid DNA Extraction Using Alkaline Lysis Method

Plasmids can be isolated by a variety of methods many of which rely on the differential denaturation and reannealing of plasmid DNA compared to chromosomal DNA. One commonly used technique developed by Birnboim and Doly involves alkaline lysis. This method essentially relies on bacterial lysis by sodium hydroxide and sodium dodecyl sulfate (SDS), followed by neutralization with a high concentration of low-pH potassium acetate. This gives selective precipitation of the bacterial chromosomal DNA and other highmolecular-weight cellular components. The plasmid DNA remains in suspension and is precipitated with isopropanol.

96-Well DNA Isolation Method From Leaf

The following protocol provides a high throughput, low cost method of producing a superior DNA yield of high quality which is suitable for TILLING, map based cloning or any application which requires long term DNA storage. The protocol has been designed for DNA extraction from leaf material, preferably young leaf tissue should be used as this minimizes samples being contaminated with polysaccharides and phenolics.

Materials:

Extraction Buffer

1. 200 mM Tris-HCL pH 7.5
2. 250 mM NaCl
3. 25 mM EDTA
4. 0.5% SDS

TE

1. 10 mM Tris-HCl pH 8.0
2. 0.1 mM EDTA pH 8.0

TE and RNAse A

• For 2 plates: 20 ml TE and 20μl DNAse-free RNase A (10 mg/ml)

The Methods are:

1. Pre-heat extraction buffer to 65°C.
2. Label collection tubes (Qiagen Cat. No. 19560) and add a single tungsten carbide bead (Qiagen Cat no. 69997) to each tube.
3. Prepare ice bucket and tube of ethanol to wash forceps after each harvest.
4. Harvest material (3 growing tips) into a single collection tube.
5. Add 400 microliters of extraction buffer to each tube (use a multi-pipettor). Put on lids (Qiagen Cat. No. 19566).
6. Homogenise material on the mixer mill (Retsch MM300) for 2 min/30s.
7. Incubate at 65^oC for 30 min to 1 hour.
8. Centrifuge for 10 minutes at full speed.
9. Label a new rack of collection tubes.
10. Remove 300 microliters of supernatant into a new collection tube (use a multi-pipettor set speed to slow) with extended length tips.
11. Carefully add 200 microliters of phenol:chloroform to each tube (THIS PROCEEDURE SHOULD BE CARRIED OUT IN THE FUME HOOD), use 200 microliters manual multi-pipettor with filter tips
12. Put lids onto the tubes and invert several times, so the samples are well mixed. Centrifuge for 10-15 minutes.
13. Label a set of storage plates.
14. Using a manual multi-pipettor with filter tips very carefully remove 200 microliters of the upper layer to a new storage plate (AB Gene Cat. No. AB 0765) (THIS PROCEEDURE SHOULD BE CARRIED OUT IN THE FUME HOOD).
15. Using a multi-pipettor add 1/10^th vol. of 3 M sodium acetate ( approximately 20 microliters) and an equal volume of isopropanol (approximately 220 microliters). Put on lids, mix well, and leave at -20^oC for a maximum of 1 hour.
16. Leave plates on desk until the have reached room temperature, as spinning when still frozen at high speeds can cause the plate to crack. Centrifuge at 5600 rpm for 45 minutes.
17. Remove supernatant and add 100 microliters of TE containing RNAse A at a final concentration of 10 microliters/ml and incubate 30 min at 37^oC.
18. Repeat the precipitation step (step 15). After centrifugation at 5600 rpm for 45 minutes remove the supernatant and add 200 microliters 70% ethanol. Put on lids and leave for 15 minutes or overnight.
19. Remove ethanol and leave to air dry. Add 100 microliters of TE and store in fridge/freezer.

Kindly Regards.


Reference: Number 17 in References

RNA Extraction From Fresh Blood

RNA can be extracted from blood since whole blood contains nucleated white cells that constitute an easily accessible source. RNA extraction from blood will be more successful if the nucleated white cells are first isolated from the red cells since the red cells are a rich source of ribonucleases that are able to degrade RNA. It is important to minimize degradation by following the appropriate recommendations for handling RNA. The methods of RNA extraction usually comprises of three steps which are cell lysis, partitioning of RNA into a solvent fraction, and recovery of RNA from the solvent by precipitation.

DNA Extraction From Fresh Bone

You can extract nucleic acids, such as DNA, from bone samples in order to analyze gene expressions, to look for somatic mutations of tumors or other pathological tissue, or for genotyping archive material when other sources of DNA are not available. You can use several kits that have already provided by biotech companies. But, if you are extracting DNA from large number samples, you can use a homemade method as described here to be effective in cost.

Cleave DNA Using Restriction Endonulease, A Bacterial Enzyme

In order to manipulate DNA you have to posses the ability to cleave DNA at specific sites by using bacterial enzyme, which is restriction endonulease. Restriction endonucleases are bacterial enzymes that cleave duplex DNA at specific target sequences with the production of defined fragments. The name of the enzyme (such as BamHl, EcoRl, AluI, and so on) tells us about the origin of the enzyme but does not give us any information about the specificity of cleavage. The recognition site for most of the commonly used enzymes is a short palindromic sequence, usually either 4, 5, or 6 bp in length, such as AGCT (for AZul),GAATTC (for EcoRl), and so on. Each enzyme cuts the palindrome at a particular site, and two different enzymes may have the same recognition sequence but cleave the DNA at different points within that sequence.

Agarose Gel Electrophoresis of Nucleic Acids

After amplify DNA template using PCR method, now you can continue your work by using gel electrophoresis in a gel composed of agarose in order to separate DNA fragments based on its molecular weight. The percentage of agarose used depends on the size of fragments to be resolved. In general a 0.8-1% gel may be used for effective separation of DNA fragments of 100-1500 base pairs.

Bacterial DNA Extraction for Pulsed-field Gel Electrophoresis

Pulsed-field gel electrophoresis (PFGE) is a method used to separate large DNA fragments such as those obtained after digestion with restriction endonucleases that cut infrequently. To avoid possible risks of shearing bacterial DNA during the extraction and digestion steps, bacterial cells are immobilized prior to processing by incorporation in agarose gel. DNA extraction for PFGE is characterized by the need to prolong contact between agarose plugs and the lysis solution that must be distributed throughout the gel. However, the duration of DNA preparation has been shortened since the initial description of the method. The method described in this posting works well with Gram-positive and Gram-negative rods of clinical interest.

Polymerase Chain Reaction (PCR): Basic Principle

Polymerase chain reaction (PCR) is a primer mediated enzymatic amplification of specifically cloned or genomic DNA sequences. PCR process was invented by Kary Mullis and it has been automated for routine use in laboratories worldwide. The main purpose of the PCR process is to amplify template DNA using thermostable DNA polymerase enzyme which catalyzes the buffered reaction in which an excess of an oligonucleotide primer pair and four deoxynucleoside triphosphates (dNTPs) are used to make millions of copies of the target sequence.

The Measurement of Tryptophan Content by Using UV-Spectrometer

The absorption of protein solutions in the UV is the result of tryptophan and tyrosine (and to a very minor, and negligible, extent phenylalanine and cysteine). The absorption maximum will depend on the pH of the solution, and spectrophotometric measurements are usually made in alkaline solutions. Absorption curves for tryptophan and tyrosine show that at the points of intersection, 257 and 294 nm, the extinction values are proportional to the total tryptophan + tyrosine content. Measurements are normally made at 294.4 nm, since this is close to the maximum in the tyrosine curve, and in conjunction with the extinction at 280 nm, the concentrations of each of the two amino acids may be calculated. This is the method of Goodwin and Morton.

Bicinchoninic Acid (BCA) Method: A Protein Assay

The Bicinchoninic acid (BCA) assay first was described by Smith, et al. BCA assay is similar to Lowry assay since it also depends on the conversion of Cu(2+) to Cu(+) under alkaline conditions. The Cu(+) is then detected by reaction with BCA. The reaction results in the development of an intense purple color with an absorbance maximum at 562 nm. BCA method and Lowry are of similar sensitivity, but BCA method is more advantageous compared to Lowry in a few things, here are the advantages:

Protein Assay using UV-Spectrophotometer at 280 nm

It is possible to estimate protein concentration in a solution by using simple spectrometer. Absorption of radiation in the near UV (280 nm) by proteins depends on the Tyrosine and Tryptophan content (also to a very small extent on the amount of Phenylalanine and disulfide bond).

Surfactant Properties: How to Quantitatively Measure

In order to measure the properties of surfactant, we can use the surface tension, emulsification activity, and hemolytic activity as the parameters. Here are the methods:

DNA Extraction Using Prepman Ultra

In my previous posting I’ve slightly given the bacterial DNA extraction method using Instagene matrix. In this particular posting, I want to give you the more simple method to extract DNA, which is using Prepman Ultra. According to the producer, Applied Biosystem, Prepman ultra is applicable to successfully preparing DNA template from bacteria, yeast, filamentous fungi, both from a plate or from tissue smears.

PCR Components

The Polymerase Chain Reaction (PCR) created a revolution in molecular biology research and its applications. PCR is an in vitro method that enzymatically amplifies specific DNA sequences using oligonucleotide primers that flank the region of interest in the target DNA. The principle involves a repetitive series of cycles each of which consist of template denaturation, primer annealing, and extension of the annealed primers by a DNA polymerase to create the exponential accumulation of a specific fragment whose ends are determined by the 5’ ends of the primers. The PCR is so named because it involves a polymerase and the products synthesized in each cycle can serve as templates in the next so the number of DNA copies approximately doubles at every cycle to create a chain reaction similar to the principles in a nuclear reactor.

Bradford Method: Colorimetric Protein Assay

Bradford method is a common colorimetric method to determine protein concentration in a sample solution. The Bradford method of protein determination is based on the binding of a dye, Coomasie Blue G, to the protein. This binding shifts the absorbtion maximum of the dye from red to blue. The absorbance of the solution is measured at 595 nm and is proportional to protein concentration when compared to a standard curve. Two types of assay are described here: the standard assay, which is suitable for measuring between 10 and 100 microgram of protein, and the microassay, which detects between 1 and 10 microgram of protein.