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:7/3/2015)... -- Pomerantz LLP announces that a class action lawsuit has ... "Company")(NYSE: PBYI ) and certain of its officers.   ... Central District of California , and ... consisting of all persons or entities who purchased Puma ... inclusive (the "Class Period").  This class action seeks to ...
(Date:7/2/2015)... ELLYN, Ill. , July 2, 2015 ... Colton Eakins are gaining invaluable real-world experiences ... of Technology this summer. The students will ... Dr. Jeff Wereszczynski,s research group at IIT. The ... Dr. Tom Carter , Physics; Dr. Kathy ...
(Date:7/2/2015)... ... July 02, 2015 , ... Sleepless nights will become obsolete ... of sleep, launched its Indiegogo campaign on June 23. Sound sleepers and the sleep ... makes it effortless to use, while the integration of the Bluetooth speaker, alarm clock ...
(Date:7/1/2015)... and PARIS , July ... and Genective, key developer of biotech crops, today ... of new generations of insect control traits. ... to counter the realities of advancing insect resistance. ... AgBiome,s unique insect control technology with Genective,s capability ...
Breaking Biology Technology:SHAREHOLDER ALERT: Pomerantz Law Firm Reminds Shareholders with Losses on their Investment in Puma Biotechnology, Inc. of Class Action Lawsuit and Upcoming Deadline - PBYI 2SHAREHOLDER ALERT: Pomerantz Law Firm Reminds Shareholders with Losses on their Investment in Puma Biotechnology, Inc. of Class Action Lawsuit and Upcoming Deadline - PBYI 3SHAREHOLDER ALERT: Pomerantz Law Firm Reminds Shareholders with Losses on their Investment in Puma Biotechnology, Inc. of Class Action Lawsuit and Upcoming Deadline - PBYI 4COD Students Participate in Prestigious Summer Internships 2Luzi Smart Lamp Recently Launched Indiegogo Campaign 2Luzi Smart Lamp Recently Launched Indiegogo Campaign 3AgBiome and Genective collaborate to create novel insect-resistant crops 2
... December 11 Olympus has further,cemented its position as ... signing an exclusive and immediate deal to distribute the ... the sole global,provider of ports for state-of-the-art laparoscopic operative ... Olympus is now the exclusive,distributor for the full range ...
... PPHM ) today announced that Thomas A. Waltz, M.D., ... Dr. Waltz had been a director of Peregrine since 2004 ... death, Dr. Waltz served on Peregrine,s Audit Committee, Compensation Committee and ... "The sudden loss of our business colleague and friend Tom Waltz ...
... Biotech Inc. ("Oncolytics") (TSX:ONC, NASDAQ: ONCY ) ... statistical endpoint in its multi-centre Phase 2 clinical ... in patients with various sarcomas that have metastasized ... of patients on study with significant, durable, clinically ...
Cached Biology Technology:Minimally Invasive Surgery: Olympus Further Enhances Credentials by Entering Into Exclusive Sales and Distribution Partnership With Market and Technology Leader Advanced Surgical Concepts 2Peregrine Pharmaceuticals Mourns Loss of Chairman Thomas A. Waltz 2Oncolytics Biotech Inc.'s REOLYSIN(R) Exceeds Primary Statistical Endpoint in U.S. Phase 2 Sarcoma Study 2Oncolytics Biotech Inc.'s REOLYSIN(R) Exceeds Primary Statistical Endpoint in U.S. Phase 2 Sarcoma Study 3Oncolytics Biotech Inc.'s REOLYSIN(R) Exceeds Primary Statistical Endpoint in U.S. Phase 2 Sarcoma Study 4
(Date:6/25/2015)... , June 25, 2015  TAKE Solutions Ltd., a ... by the United States Patent and Trademark Office (USPTO) ... process leverages TAKE Solutions, Clinical Accelerators to reduce the ... (when compared to standardization without the accelerators), thus reducing ... At the heart of the patented ...
(Date:6/24/2015)... -- Research and Markets( http://www.researchandmarkets.com/research/hhwlfj/huawei_ascend ... "Huawei Ascend Mate 7 Fingerprint Sensor - Reverse ... Huawei,s technological choice for the fingerprint sensor ... and Samsung,s one. The Ascend Mate 7 presents ... a main actor in capacitive sensing human interface ...
(Date:6/23/2015)... RALEIGH, N.C. , June 23, 2015 /PRNewswire/ ... technology, today announced the results of a recent ... unmatched accuracy on the wrist during activity. In ... biometric technology was compared along with the Apple ... for accurately measuring heart rate during activity. The ...
Breaking Biology News(10 mins):TAKE Solutions Awarded Patent By USPTO 2Huawei Ascend Mate 7 Fingerprint Sensor - Reverse Costing Analysis 2Valencell Delivers Breakthrough Heart Rate Accuracy on the Wrist 2Valencell Delivers Breakthrough Heart Rate Accuracy on the Wrist 3
... ) can today be found all over Central Europe, Asia, ... in the Iberian Peninsula at the end of the Early ... an international research study, with Spanish participation, which has discovered ... of fossils found in the Cueva Victoria deposit in Cartagena ...
... of newly installed, hands-free faucets at The Johns Hopkins Hospital, ... hands and dispense preset amounts of water, shows they were ... common and hazardous bacteria in hospitals compared to old-style fixtures ... "Newer is not necessarily better when it comes to ...
... (SACRAMENTO, Calif.) UC Davis cancer researchers have found ... for surveillance with regular CT scans over lymph node ... findings, published online last week in the journal Cancer, ... cancers with surveillance when discussing treatment options with their ...
Cached Biology News:Green toad inhabited Iberian Peninsula 1 million years ago 2Latest hands-free electronic water faucets found to be hindrance, not help, in infection control 2Latest hands-free electronic water faucets found to be hindrance, not help, in infection control 3Latest hands-free electronic water faucets found to be hindrance, not help, in infection control 4Frequent CT scanning for testicular cancer surveillance associated with secondary malignancies 2Frequent CT scanning for testicular cancer surveillance associated with secondary malignancies 3
VACUGENE PUMP 110V, 1 EA. Category: Nucleid Acid Detection Systems....
Rat whole blood (Sprague-Dawley, averaging 7-8 weeks of age, either sex) is collected at the time of slaughter and refrigerated within 6 hours, and processed and frozen within 24 hours. The serum is ...
... BMH 71-18 mutS and ES1301 mutS ... coli. Use of these strains prevents repair ... leading to high mutation efficiencies and making ... GeneEditor and Altered Sites II Mutagenesis Systems. ...
propidium iodide *1.0 mg/mL solution in water*...
Biology Products: