Navigation Links
Preparation of PCR Quality Template DNA from a Wide Range of Tissues Using InstaGene Matrix

Paul Burr, Wellcome Clinical Scholar, Department of Veterinary Pathology, University of Glasgow, Bearsden Rd, Glasgow. G61 1QH.


Introduction
A method that can be conveniently used to prepare DNA from a wide range of tissues for analysis in the Polymerase Chain Reaction is described. The method is both quick and simple, with a low risk of inadvertent contamination of samples due to the reduced number of manipulations required compared to other DNA extraction methods.


Methods
Canine tissues (tri-geminal ganglion, lumbo-sacral ganglion, brain, mesenteric lymph node, sub-mandibular lymph node, tonsil, sub-mandibular salivary gland, parotid salivary gland, spleen, liver and kidney) were collected from a number of dogs during routine post mortem. All tissues were collected within 2 hours of death and frozen at -70 C until processed. Great care was taken during preparation to avoid problems of both environmental and cross contamination.

1. Cut a small (3 mm3) piece of tissue on a fresh glass slide using a fresh scalpel blade. Macerate slightly with the blade and place in a sterile 1.5 ml screw cap Eppendorf tube.

2. Add 1 ml of 1x Trypsin/EDTA (Gibco BRL). Incubate at room temperature for 15-30 minutes, shaking occasionally.

3. Spin down (13K, 2-3 minutes) to pellet tissue, and carefully remove all the supernatant.

4. Add 250 l of InstaGene matrix (Bio-Rad Laboratories), and mix well. Incubate at 56 C for 30 minutes, mixing occasionally.

5. Vortex tube thoroughly for 10 seconds, place in boiling water bath or 100 C heat block for 8 minutes.

6. Vortex thoroughly for 10 seconds, spin at 13K for 2-3 minutes. Use 20 l of the supernatant for each PCR reaction.

Notes: Store remainder at -20 C for subsequent PCRs. Repeat step 6 when reusing the preparation (allow to thaw first).


PCR
Primers used in this experiment were designed to amplify a 120 base pair fragment of the Canine Pancreatic Lipase Gene (Mickel et al., 1989). Amplification was carried out using 2 Units AmpliTaq DNA polymerase (Perkin-Elmer) mixed with an equal volume of TaqStart antibody (Clontech) per reaction in a total reaction volume of 50 l, containing 50 pmol each primer and 200 mol/L of each deoxynucleoside triphosphate in 10 mM Tris (pH 8.4), 50 mM KCl, and 1.5 mM MgCl2. Thermal cycling was performed on a Perkin-Elmer 9600. Initial denaturation was at 94 C for 5 minutes, followed by 40 cycles of 94 C for 30 seconds, 57.5 C for 1 minute, then a final extension of 72 C for 7 minutes.


Results

All tissues examined produced a clear band of the correct size. The variation in the brightness of the band does not appear to correspond to the amount of DNA template present in each reaction. Apart from the positive control (A72 cell line), there are two template copies per cell for each tissue. The most likely cause of the variation in intensity of the band obtained is therefore inhibitory factors present within the sample. It is important not to exceed the capacity of the InstaGene matrix by using too much tissue in the initial preparation. Exceeding the matrix capacity may cause PCR-inhibitory factors normally removed by the procedure to remain in the supernatant. Carryover of these inhibitory factors may affect amplification in the subsequent PCR reaction.


Conclusion
InstaGene matrix can be used to rapidly prepare PCR compatible DNA from a wide range of tissues in about 2 hours. Preparation and amplification can be carried out in the same day if required.


Acknowledgements
This work was supported by a grant from The Wellcome Trust.


References
Binn, L. N., Marchwicki, R. H. and Stephenson, E. H., Establishment of a Canine Cell Line: Derivation, Characterization, and Viral Spectrum, Am. J. Vet. Res. 41, 855-860 (1980).

Mickel, F. S., Weidenbach, F., Swarovsky, B., Laforge, K. S. and Scheele, G. A., Structure of the Canine Pancreatic Lipase Gene, J.Biol.Chem., 264, 12895-12901 (1989).


* The Polymerase Chain Reaction (PCR) process is covered by patents owned by Hoffmann-LaRoche. Use of the PCR process requires a license.

Eppendorf is a trademark of Eppendorf Geratebau netheler + Hinz, GmbH.


back to top
'"/>

Source:


Page: All 1 2 3

Related biology technology :

1. Sample Preparation for Microinjection
2. High Pure PCR Template Preparation Kit
3. Proteinase K, Recombinant, PCR Grade: the Ideal Tool for Template Preparations
4. Dicer vs. RNase III for Preparation of siRNA Cocktails
5. Genomic DNA Preparation from RNAlater Preserved Tissues
6. Commercial Plasmid Preparation Methods Yield DNA With Different Transfection Capabilities
7. Sample Preparation Solutions for Cellular Protein Fractionation Poster, Rev A
8. High Throughput Plasmid DNA Preparation Using A Magnetic Bead Kit On An Automated Workstation
9. A Simple and Rapid Technique to Purify High-Quality Midiprep DNA
10. Generate High-Quality, Directional Plasmid cDNA Libraries
11. Quality control of antibodies using the 2100 bioanalyzer and the Protein 200 Plus assay
Post Your Comments:
(Date:11/22/2014)... MD (PRWEB) November 21, 2014 ... management solutions provider, has released Limfinity® version 5.1.4. ... installation projects of Limfinity® than ever in RURO’s ... configuration and minority framework enhancement. Limfinity® version 5.1.4 ... far above and beyond bug fixes! , Limfinity® ...
(Date:11/22/2014)... Quebec , November 21, 2014 ... wechselt als Chief Commercial Officer zu KLOX ... SVP der Unternehmensentwicklung und Chief Financial Officer bestellt    ... Europa mit der Vermarktung seines jüngst zugelassenen Behandlungssystems zur ... Inc. ("KLOX" bzw. das "Unternehmen") ist sehr erfreut, die ...
(Date:11/21/2014)... Angeles, CA (PRWEB) November 20, 2014 ... “A Short Walk to the Other Side” published ... an “Award-Winning Finalist in the “Fiction: Short Story” category ... , Jeffrey Keen, President and CEO of USA Book ... from mainstream and independent publishers, including Simon & Schuster, ...
(Date:11/21/2014)... 2014 Why did Stephen Hawking become ... so instantly recognizable? Why have they become icons to ... author Hilton Ratcliffe seeks out the answers to those ... to do with science. In " Stephen Hawking Smoked ... 21, 2014), Ratcliffe puts it plainly: , “It ...
Breaking Biology Technology:RURO Announces Limfinity® version 5.1.4 2KLOX Technologies meldet Neubesetzung von Führungspositionen 2KLOX Technologies meldet Neubesetzung von Führungspositionen 3KLOX Technologies meldet Neubesetzung von Führungspositionen 4KLOX Technologies meldet Neubesetzung von Führungspositionen 5Mystic Ink 's Short Story Collection “A Short Walk to the Other Side” Honoring Ray Bradbury Praised As Award-Winning Finalist In USA Best Book Awards 2Mystic Ink 's Short Story Collection “A Short Walk to the Other Side” Honoring Ray Bradbury Praised As Award-Winning Finalist In USA Best Book Awards 3Mystic Ink 's Short Story Collection “A Short Walk to the Other Side” Honoring Ray Bradbury Praised As Award-Winning Finalist In USA Best Book Awards 4“Stephen Hawking Smoked My Socks” Sparks Controversy in Science World 2“Stephen Hawking Smoked My Socks” Sparks Controversy in Science World 3“Stephen Hawking Smoked My Socks” Sparks Controversy in Science World 4
... Wis. Researchers led by James Dahlberg, a founder ... found that a super-small genetic element may play a critical ... ,Teams led by Dahlberg and collaborator Wayne Tam at the ... reported this week in the Proceedings of the National Academy ...
... University of Wisconsin-Madison has been assigned 65,000 Internet addresses ... forward with a planned expansion of its wireless network ... body, the American Registry for Internet Numbers , ... pushing 80 percent usage of its current allocation. The ...
... dose of national honors on Monday as President Bush ... R. de Boor and the Wisconsin Alumni Research ... respectively. , ,The 2003 National Medals of Science ... They included discoveries about how genes work, the invention ...
Cached Biology Technology:Third Wave co-founder shoots for small target in latest project 2UW-Madison wireless plan requires more net addresses lots more 2
(Date:11/11/2014)... , Nov. 11, 2014  Forensicon, Inc., a ... is pleased to announce the promotion of Yaniv ... of Digital Forensics. In Schiff,s new role as Director, ... team of digital forensics examiners and provide leadership within ... Photo - http://photos.prnewswire.com/prnh/20141110/157719 Schiff ...
(Date:11/7/2014)... , Nov. 7, 2014  In conjunction with the ... Police Departments, The Community Foundation of the Verdugos has announced ... DNA JUSTICE PROJECT," a $250,000 initiative to keep the Verdugo ... In its first six months of existence, the Lab has ... support for local law enforcement. "Because our number ...
(Date:11/6/2014)... deaths occur because of metastasis, yet progress in ... slow. , "It,s been particularly challenging to design ... research fellow in systems biology at Harvard Medical ... until after they,ve already metastasized." , Gujral and ... should help researchers better understand how metastasis begins. ...
Breaking Biology News(10 mins):Yaniv Schiff Promoted to Director of Digital Forensics at Forensicon, Inc. 2Community Foundation Of The Verdugos Unveils Campaign To Fund Glendale-Burbank DNA Justice Project 2Migration negation 2Migration negation 3
... 2014The ability to reprogram adult cells so they return ... cellis enabling the development of promising new cell therapies. ... factors that promote pluripotency, such as the Brg1 protein ... Open Access , a peer-reviewed journal from Mary Ann ...
... and around Snowy River National Park in Victoria, Australia were ... Aqua satellite on February 06, 2014. Actively burning areas, detected ... dry, windy conditions in Victoria have been responsible for the ... fires in this image are located in and around the ...
... health, according to new research. A study from researchers at ... your spouse is supportive and how you support your ... cardiovascular health. The findings reveal that when both partners ... that is, sometimes helpful and sometimes upsetting each partner,s ...
Cached Biology News:Heart disease risk linked with spouses' social support 2
... siRNA --- a cocktail of 3 siRNAs ... interestis our most popular product. The siTrio ... knockdown of your target gene when used ... transfection and confirmation of optimal transfection with ...
...
... MULTI-FUN Incubator is a compact benchtop ... a microprocessor controller with a large ... and agitation speed. Air circulation inside ... incubator. Temperature can be easily calibrated ...
... is a compact benchtop and stackable ... controller with a large display screen ... speed. Air circulation inside to evenly ... can be easily calibrated from the ...
Biology Products: