Tuesday, 28 May 2013

LAB 6 : EXTRACTION AND MEASUREMENT OF PLASMID DNA USING GF-1 PLASMID DNA EXTRACTION KIT


INTRODUCTION
Every living cell contains DNA material that coded for its characteristic such us colour and cell function. There are several methods to isolate the DNA. DNA isolation is a routine procedure to collect DNA for subsequent molecular or forensic analysis. There are three basic and two optional steps in a DNA extraction breaking the cells open, commonly referred to as cell disruption or cell lysis, to expose the DNA within. This is commonly achieved by chemical and physical methods-blending, grinding or sonicating the sample, removing  membrane lipids by adding a detergent or surfactants, removing proteins by adding a protease (optional but almost always done), removing RNA by adding an RNase (often done) and precipitating the DNA with an alcohol — usually ice-cold ethanol or isopropanol. Since DNA is insoluble in these alcohols, it will aggregate together, giving a pellet upon centrifugation. This step also removes alcohol-soluble salt.
Plasmids, which are small molecules of DNA that can replicate independently of the chromosomal DNA. They are double-stranded and, in many cases, circular. Plasmids usually occur naturally in bacteria, but are sometimes found in eukaryotic organisms.
          Plasmid is used in recombinant DNA experiments to clone genes from other organisms and make large quantities of their DNA. Plasmid can be transferred between same species or between different species. Size of plasmids range from 1-1000 kilo base pairs. Plasmids are part of mobilomes (total of all mobile genetic elements in a genome) like transposons or prophages and are associated with conjugation. Even the largest plasmids are considerably smaller than the chromosomal DNA of the bacterium, which can contain several million base pairs.
Extraction is an easy and quick way to purify DNA from a mixture of proteins, lipids and nucleic acids.  It is important to keep in mind the purpose of this procedure while performing it. The reason for this procedure is to separate the plasmid DNA from its associated proteins so that further manipulations can be done to it. Enzymes added to purify DNA in vitro can have unhindered access to it. These enzymes might be used for restriction mapping, ligation, sequencing, or other procedures to modify the DNA. A poorly purified plasmid preparation will only be partially accessible to the enzymes and this will cause many headaches in these later steps. For this reason, special care must be taken to ensure a pure DNA preparation.
A diphenylamine (DPA) indicator will confirm the presence of DNA. This procedure involves chemical hydrolysis of DNA: when heated (e.g. ≥95 °C) in acid, the reaction requires a deoxyribose sugar and therefore is specific for DNA. DNA concentration can be determined measuring the intensity of absorbance of the solution at the 600 nm with a spectrophotometer and comparing to a standard curve of known DNA concentrations. Measuring the intensity of absorbance of the DNA solution at wavelengths 260 nm and 280 nm is used as a measure of DNA purity. DNA absorbs UV light at 260 and 280 nanometres, and aromatic proteins absorb UV light at 280 nm; a pure sample of DNA has a ratio of 1.8 at 260/280 and is relatively free from protein contamination. A DNA preparation that is contaminated with protein will have a 260/280 ratio lower than 1.8.

OBJECTIVE
To understand and practice proper technique of isolation process of plasmid DNA from bacteria.

MATERIALS AND REAGENTS
Centrifuge
Pipette
Incubator ( Digital Dry Bath )
Column
Microcentrifuge tube
Ethanol
Elution Buffer
Wash Buffer
Proteinase K
Buffer R1 and R2




PROCEDURES

Reminder :
* All steps are to be carried out at room temperature unless stated otherwise.
* Wash Buffer (concentrated) has to be diluted with absolute ethanol before use. Please
   refer to Reconstitution of Solutions .
* If precipitation forms in Buffer BG , incubate at 55°c - 65°c with occasional mixing until
   completely dissolved.

1. Centrifugation


1 mL of bacteria culture grown overnight or culture grown to log phase formed pellet    by centrifugation at 6000  x g for 2 min at room temperature. Supernatant was decanted completely.
Through removal of supernatant is essential as residual culture media may affect both yield and purity.




2. Re-suspension of pellet
100µL Buffer R1 was added to the pellet and the cells were re-suspended completely by pipetting up and down.
Ensure complete cell re-suspension. Lysis will not occur if clumps of bacteria remain following an inefficient re-suspension procedure.



3.Lysozyme treatment
10 µL lysozyme (50mg/mL) was added into the cell suspension of Gram-negative bacteria strains. While, for Gram-positive bacteria strains , 20 µL lysozyme (50mg/mL) was added into the cell suspension. They were then mixed thoroughly by vortex and were incubated at 37°C for 20 min.
Some bacteria strains may require longer incubation time in lysozyme.

4.Centrifugation
Pellet was formed when digested cells go through centrifugation at 10000 x g for 3 min. Supernatant was being decanted completely.



5.Protein denaturation
Pellet in 180 µL of Buffer R2 was re-suspended and 20 µL of Proteinase K was added. Vortex was used to mix thoroughly. It was then incubated at 65°C for 20 min with occasional mixing every 5 min.
Lysate should be clear at the end of incubation or else extend the incubation time to 30 min.

6.Homogenization
400 µL of Buffer BG which without RNase A treatment was added and mixed thoroughly by inverting tube several times until a homogeneous solution is obtained. It was incubated for another 10 min at 65°C.

 7.Addition of Ethanol
200 µL of absolute ethanol was added. The sample was mixed up immediately and thoroughly.
Mix immediately to prevent uneven precipitation of nucleic acid due to high local ethanol concentrations.

8.Loading to column
The sample was transferred into a column assembled in a clean collection tube.  Centrifugation at 10000 x g for 1 min was needed. After that, flow through was discarded.
In order to obtain maximum yield, we fix the orientation of the column during centrifugation at all time. We place the column which has a triangle mark on the edge, at a fixed position during centrifugation.
If column clogs, add 200 µL Buffer BG into column and centrifuged it.

9.Column washing
The column with 750 µL of Wash Buffer was washed and centrifuged at 10000 x g for 1 min. Flow through was also discarded.
Ensure that ethanol has been added into the Wash Buffer before use.

10.Column drying
The column undergo centrifugation at 10000 x g for 1 min to remove residual ethanol.
This step has to be carried out to remove all traces of ethanol as residual ethanol can affect the quality of DNA and may subsequently inhibit enzymatic reactions.

11.DNA elution
The column was placed into a clean micro-centrifuge tube. 100 µL of preheated Elution Buffer was added directly onto column membrane and stand for 2 minutes. DNA was eluted by centrifuged it at 10000x g for 1 minute and stored at 4°C or -20°C.
Ensure that the Elution Buffer is dispensed directly onto the center of the membrane for complete elution.   







RESULTS


DISCUSSIONS

There are some precaution steps to take when we are doing the experiment :
1) When re-suspend the cell completely by pipetting up and down, place the pipette on the same position to make 
    sure complete cell re-suspension.
2) When centrifuge , place the column which has a triangle mark on the edge to ensure that the process achieve
    optimization.


Problem Probability Suggestions
Low DNA yield Incomplete cell re-suspension Ensure that cells are completely re-suspended in Buffer R1, and Buffer R2 before incubation in lysozyme and Proteinase K.
Low elution efficiency Preheat Elution Buffer to 65 oC - 75 oC before eluting DNA
Low purity Incomplete protien denaturation Ensure that cells are completely re-suspended in Buffer R1, and Buffer R2 before incubation in lysozyme and Proteinase K. Extend incubation time until lysate clears.
RNA contamination Add RNase A to the sample as indicated in the protocol. Ensure that Rnase A used has not been repeatly frozen thawed. If necessary prepared a fresh stock.
Difficult to re-suspend cell pellet in Buffer R1 or R2 Centrifugation at high speed and long periods Ensure that the cell culture is centrifuged at the recommended speed and time.
Using pipette tips ,pipette the lysate up and down until completely homogenized.
Poor performance of eluted DNA in downstream application Eluted DNA contents traces of ethanol Ensure that the Column drying step is carried out prior to elution.

CONCLUSION

DNA is extracted from human cells for a variety of reasons. With a pure sample of DNA you can test a newborn for a genetic disease, analyze forensic evidence, or study a gene involved in cancer. Therefore, the practice on DNA extraction and measurement should be carried out carefully and precaution should be taken in order to get a pure DNA.

REFERENCES



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