Lab6: Extraction of Plasmid DNA using GF-1 Plasmid DNA extraction Kit

Introduction

          The GF-1 Plasmid DNA Extraction Kit is designed for rapid and efficient purification of high copy and low copy plasmid DNA from bacterial lysates. This it uses a specially-treated glass filter membrane fixed into a column to efficiently bind DNA in  the presence of high salt. Combining alkaline  lysis-SDS and  minicolumn spin technology, up o 20μg of plasmid DNA  from bacterial cultures can be isolated.This kit uses  the alkaline lysis-SDS method to lyse cells and release  plasmid DNA.Special buffers provided in the kit are optimized to enhance binding of DNA onto  a specially-treated  glass filter  membrane for efficient  recovery of highly pure plasmid DNA.

          The purification is based on the usage of denaturing agents to provide efficient cell lysis, denaturation of proteins and subsequent release of DNA. Special buffers provided in the kit are optimized to enhance binding of DNA onto a specially-treated glass filter membrane for efficient recovery of highly pure plasmid DNA

Plasmids are double-stranded circular DNA molecules that have the property of self-replication, independent of chromosomal DNA. Although the presence of a plasmid in a bacterial cell may be detected genetically as a change in phenotype, often it is necessary to isolate plasmid DNA for molecular studies, such as size determination, restriction enzyme mapping, and nucleotide sequencing, or for the construction of new hybrid plasmids. The degree of purification required will depend upon the intended use.

          In the process, the modified alkaline lysis method and RNase treatment are used to get cleared cell lysate plasmid DNA in crude lysate has been bound to the column; the contaminants can be washed off with wash buffer. Finally, the purified plasmid DNA is eluted by a high salt buffer and then precipitated with isopropanol for desalting. The entire procedure can be completed in 120minutes without ultracentrifuges and HPLC or other toxic reagents.

Storage Conditions
Plasmid DNA Extraction Kit can be stored at room temperature (15-25 ℃). After adding RNase A, PDE Buffer1, should be stored at 4 ℃ and is stable for six months. Other buffers and columns can be stored dry for up to 1 year at room temperature (15-25 ℃)

Chemical Hazard

Buffer NB contains guanidine salts which can be harmful when in contact with skin orswallowed.

Always wear gloves and practice standard safety precautions. Do NOT disinfect guanidine orextraction waste in solutions containing bleach or any other form of acid. To clean any itemscontaminated with the reagent, simply soak in detergent and water to remove all traces ofguanidine before cleaning with bleach or acidic solutions.

Result

OD230 = 0.229

OD260 = 0.371

OD280 = 0.178

Ratio (OD260/ OD280)

= (0.371 ÷ 0.178)

= 2.084

Ratio (OD260/ OD230)

= (0.371 ÷ 0.229)

= 1.620

OD260 = 0.371

DNA concentration (µg/mL)

= 50 µg/mL x OD260 x dilution factor

= 50 x 0.371 x 50

= 927.5µg/mL

Total yield in 500µL sample

= DNA concentration x volume of sample in milliliters

= 927.5 µg/mL x 0.50 mL

= 463.75 µg

Discussion

            First, the S1 added to the pellet and the cell is re-suspended completely by vortexing or pipetting. The cells are re-suspended completely because lysis will not occur if clumps of bacteria remain following an inefficient re-suspension procedure. Then, the S2 added. It mixed gently by inverting tube several times (4-6 times). Vortex could sheared the genomic DNA and leads to contamination. So, it been avoided.  Then, the tube incubated in ice for less than 5 min. It reduced non-supercoiled plasmid contamination in bacteria strains and precipitation could be carrying out more effective in the cold. Buffer NB added and mixed it gently by inverting until white precipitate form and centrifuged it. After centrifugation, the compact white precipitate been spun down and was separate from the supernatant. The supernatant was transfer into column assembled in a clean collection tube. In order to obtain maximum yield, the orientation of the column was fix during centrifugation at all the time. The column that has a triangle mark on the edge was place at a fixed position during centrifugation. After that, the column is washed with wash buffer, centrifuged and discarded the flow through. The column was centrifuge again to remove residual ethanol because the quality of DNA could be affect by ethanol. Subsequently, the enzymatic reactions are inhibited. Then, the column was place into a clean microcentrifuge tube.50 µL of Elution Buffer is added onto column membrane and stood for one minute and centrifuged it. The elution buffer is dispensed directly onto the centre of the membrane for complete elution. 490 µL of sterile water is added to dilute the mixture. Finally, the mixture was transfer in a cuvette. The absorbance at 230nm, 260nm and 280nm are measured.

            The ratio of OD260 to OD280 that we got was 2.084 that were approaching 1.80. This shown there had less contaminant with protein. If there was contaminant with protein, the OD260/OD280 will be significant less.

            Besides, the ratio of OD260/OD230 that we got was 1.620, which was greater than 1.50. This reading represent the present of organic compound or chaotropic salts in the sample. This shown the amount of salt present in the our sample were low because the greater the amount of salt, the lower the ratio.

The cells are lysed under very carefully controlled conditions, the DNA fragment are still very large , much larger than plasmid and could be removed with cell debris by centrifugation.

           The methods used to purify the plasmid DNA from the cell lysate rely on the small size and abundance of the plasmid DNA relative to the chromosome, and the covalently closed circular nature of plasmid DNA. Most plasmids existed in the cytoplasm of the cell as circular DNA molecules that are highly supercoiled.

            Note that by not adhering to the recommended protocols, unsatisfactory results related to yield and quality of DNA may occur. The table below is problems that may occur and ways to overcome it.

Problem	Possibility	Suggestions
Low DNA yield	Cell lysis incomplete / Lysate did not clear after addition of S2	Do not exceed the recommended culture volume of 5ml
		Use fresh S2 by preparing as follows: 0.2N NaOH, 1% SDS.
	Poor resuspension of cells	Ensure that cells are completely resuspended after the addition of S1. No cell clumps should be visible
	Low copy-number plasmid	Increase culture volume or grow culture in enriched medium such as Terrific Broth to increase the yield.
	Bacteria culture overgrown or not fresh	Do not culture bacteria longer than 20 hours at 37°C as this may lower the plasmid yield. Media should contain antibiotic at an appropriate concentration.
	Column not placed at fixed orientation during centrifugation	Place the column which has a triangle mark on the edge, at a fixed position during centrifugation at all times.
	Elution is not performed properly	Ensure that the elution buffer used is a low salt buffer or water with a pH range of 7.0 - 8.5.
Column clogged	Transfer of precipitate from sample prior to loading into column	Ensure that white precipitate is not transferred over during loading of column to prevent clogging up of the membrane.
High molecular weight DNA contamination.	Vigorous mixing of lysate upon addition of S2 or Buffer NB	Do not vortex or mix vigorously after addition of S2 or Buffer NB. Simply mix by gently inverting the tube a few times.
	Incubation longer than 5 min after addition of S	Do not incubate longer than 5 minutes.
Additional plasmid formation	Irreversible denaturation during cell lysis	Do not carry out incubation longer than 5 minutes after the addition of S2.
	Nicked circular plasmids due to the presence of nuclease	Carry out purification without delay at least until the washing step where nucleases will be removed.
		Incubation on ice after addition of S2 reduces nuclease activity.
RNA Contamination	RNA digestion was insufficient	Ensure that RNase A has been added into S1 or add a new preparation of RNase A into S1 to a final concentration of 100µg/ml.
Poor performance of eluted DNA in downstream applications	Eluted DNA contains traces of ethanol	Ensure that the Column drying step carried out prior to elution.
		Use Elution Buffer or water with a pH range of 7.0 - 8.5.

Conclusion

          Extraction is an easy and quick way to purify DNA from a mixture of proteins, lipids and nucleic acids (e.g., a cell or bacterial lysate). The extract is deprotenised, the RNA be removed and the DNA is concentrated by ethanol precipitation. In a plasmid preparation, the plasmid DNA from the large amount of bacteria chromosomal DNA is necessary to be separate.

          Plasmid DNA Extraction Kit was design for rapid and efficient purification of high copy and low copy plasmid DNA without the need for precipitation or organic extractions. It used a specially treated glass filter membrane fixed into a column to bind DNA efficiently in the presence of high salt. Special buffers provided in the kit were optimizing to enhance binding of DNA onto a specially treated glass filter membrane for efficient recovery of highly pure plasmid DNA. Enzymes added to purify DNA in vitro could have unhindered access to it. These enzymes could be use for restriction mapping, ligation, sequencing, or other procedures to modify the DNA.

         Multiple samples could be process rapidly and with practice, the purification takes less than 30 minutes. Optimized buffers ensure only highly pure plasmid DNA is extracted. It was ready to use in many routine molecular biology applications such as restriction enzyme digestion, radioactive/fluorescence DNA sequencing, PCR, ligation, transformation and other manipulations.

Reference

http://www.taq-dna.com/plasmid-dna-purification-_400.html

http://www.taq-dna.com/rich_files/attachments/DNA_Purification_Kits__DNA_extraction_Kits__Mini_prep_kits__DNA_Isolation/Plasmid_DNA_Extraction_isolation_purification%20miniprep_Kit.pdf

Book:

Biotechnology-4 Including recombinant DNA technology,EnvironmentalBiotechnolog,Animal cell culture by S.Mahesh and A.B Vedamurthy

Bacterial Genetics by Nancy Trun and Janine Trempy

http://www.osti.gov/energycitations/product.biblio.jsp?osti_id=5021853

http://vpqsci.ca/store/index.php?main_page=product_info&cPath=4&products_id=30

http://www.clonagen.com/clonagen/9bd22dc0-aaa0-46c3-ba51 5df7d9cdcbaa/gf_1_forensic_dna_extraction_kit_product.aspx

http://www.taq-dna.com/plasmid-dna-purification-_400.html

http://faculty.plattsburgh.edu/donald.slish/Extraction.html

http://www.favorgen.com/products_for_res_nae_faPDE_Mega.htm