Navigation Links
New Protocols for Isolating High- Molecular-Weight Genomic DNA


Rapidly recover intact DNA from blood, bone marrow, and cultured cells

Brenda J. Rogers Mark J. Dycaico
Stratagene

Stratagene now offers updated protocols to isolate and purify high-molecular-weight genomic DNA using the RecoverEase DNA isolation kit. New procedures include DNA isolation from blood, bone marrow, and tissue culture cells. In addition, protocols for liver, lung, kidney, spleen, testes, and brain are also included. Each procedure eliminates the need for toxic phenol or chloroform and requires very little hands-on time to perform. Use the kit to minimize physical manipulation of the DNA the point in purification methodology where DNA is most susceptible to shearing forces. Fully hydrated high-molecular-weight DNA is ready to use in molecular biological applications in less than 24 hours.

The RecoverEase DNA isolation kit is designed to isolate very high-molecular-weight genomic DNA by reducing the shearing forces that take place in traditional methods using phenol-chloroform extraction and ethanol precipitation. The basic kit procedure requires tissue homogenization, filtration through a filter unit supplied with the kit to remove cellular debris (<3 minutes), isolation of the cell nuclei by centrifugation (<20 minutes), and protease digestion of the nuclei (45 minutes).1,2 The resulting suspension is transferred to a free-floating RecoverEase kit cup where the digested proteins and low molecular weight contaminants are removed by dialysis (16 to 48 hours), leaving behind fully hydrated DNA, which is ready for immediate use. From previous experiments using field inversion analysis, DNA isolated in this way, produced products that were 500-kb long.2 Subsequent Southern blot analysis demonstrated that the DNA cut with a variety of restriction enzymes (data not shown). These benefits, along with higher packaging efficiencies, make the RecoverEase DNA isolation kit the recommended method for purifying high-molecular-weight genomic DNA from blood, bone marrow, and cultured cells.

Big Blue Transgenic Rodent Mutation Assay and Cell Culture System

Genomic DNA isolated with this kit is ideal for recovering lambda shuttle vectors in transgenic in vivo mutation assay systems, such as Stratagenes Big Blue mutation assay systems.*,3,4,5 An important step in these mutation assays is retrieving large quantities of shuttle vector from the animals genomic DNA for subsequent analysis of mutations within a target region of the vector. The shuttle vector in Big Blue was derived from bacteriophage lambda and can be retrieved from the animal by subjecting the genomic DNA to the specialized in vitro lambda Transpack packaging extract.**,4 Because the shuttle vector is nearly 50-kb long and is present in multiple copies within the genome of the animal, efficient packaging is contingent on genomic DNA preparations of very high-molecular weight.6 The DNA must also have sufficient purity so as to be free of substances that inhibit the packaging reaction. For these reasons, measuring packaging efficiency in DNA preparations made from shuttle vector transgenics is a good indicator of DNA size and purity. DNA yielded from using the kit averaged a 74% greater recovery of plaque-forming units (pfu) per Transpack packaging reaction over organic extracted DNA overall for liver, kidney, spleen, testes, lung, and brain in Big Blue transgenic mice.6

RecoverEase DNA Isolation Kit vs. Organic Extraction

The RecoverEase method (see Methods) was compared with the organic extraction technique in relation to DNA yield, quality, and packaging efficiency. The latter technique was taken from Stratagenes Big Blue DNA isolation kit, which uses a modified phenol-chloroform extraction and an ethanol precipitation protocol optimized for isolating high-molecular-weight genomic DNA from transgenic animals containing lambda shuttle vectors. The Big Blue DNA isolation kit was the first kit specifically designed to prepare genomic DNA of size and quality that, when subjected to in vitro packaging extracts, yielded large numbers of rescued shuttle vector.

Table 1
Average Lambda Phage Recovery with RecoverEase DNA Isolation Kit and Organic Extraction Methods

Tissue

Tissue source

Method

Average yieldb

Average pfu/reactionc

Average total pfu recoverabled

Average pfu/mg DNAe

Blood

Big Blue rats

Dialysis
(RecoverEase kit)

45 mg

226,000

3,892,000

86,000

Organic Extraction

NRf

NR

NR

NR

Bone marrow

Big Blue mice

Dialysis
(RecoverEase kit)

180 mg

898,000

29,126,000

162,000

Organic Extraction

150 mg

798,000

7,477,000

50,000

Big Blue rats

Dialysis
(RecoverEase kit)

76 mg

200,000

5,884,000

77,000

Organic Extraction

100 mg

122,000

1, 148,000

11,000

Fibroblast
(cultured cells)

Big Blue mice

Dialysis
(RecoverEase kit)

138 mg

702,000

28,417,000

206,000

Organic Extraction

102 mg

636,000

5,959,000

58,000

Big Blue
Rat 2

Dialysis
(RecoverEase kit)

96 mg

486,000

15,551,000

162,000

Organic Extraction

86 mg

419,000

3,929,000

46,000

Overall averageg

Dialysis
(RecoverEase kit)

122 mg

572,000

19,744,000

152,000

Organic Extraction

110 mg

494,000

4,628,000

41,000

a Results are the averages of 2 to 5 samples each.

bAn aliquot of the recovered DNA was sheared through an 18-gauge needle, and the optical density at 260 nm was used to determine the DNA concentration for each sample. The concentration was multiplied by the volume recovered to give the DNA yield.

cDNA (8 l) was packaged per the Transpack protocol, and an aliquot of the total DNA was plated on the Big Blue SCS-8 E. coli strain to determine the number of pfu per reaction.

dThe estimated total pfu recoverable was determined using the number of pfu per reaction multiplied by the number of estimated reactions achievable from the total volume of DNA recovered.

eThe pfu/ug DNA was determined by dividing the total recoverable pfu by the total yield of DNA.

fNR: none recovered.

g The average of all samples for mice, rats, and tissues combined (for each extraction technique). For comparison purposes, average values are for bone marrow and fibroblast only.

Blood samples were collected from Big Blue rats, while individual bone marrow samples were taken from both Big Blue mice and Big Blue rats. The Big Blue mouse embryonic fibroblast and Big Blue rat 2 embryonic fibroblast cell lines were also tested. Genomic DNA was isolated from two to five individual samples of each tissue type, using both dialysis and organic extraction methods. The results of the comparison are shown in Table 1. When using 5 to 7 ml of rat blood, the standard organic extraction protocol (Big Blue DNA isolation kit) failed to yield DNA, even after two attempts. However, other organic extraction protocols begin with a blood volume of 20 ml7 so the lack of DNA recovery may have been due to an insufficient starting blood volume. The dialysis method yielded enough high-molecular-weight DNA from the same blood volume (5 to 7 ml) to package an average of over 3 million pfu of shuttle vector per sample. Among the remaining tissues tested, packaging efficiencies averaged 16% higher with the RecoverEase DNA samples, compared to those prepared using organic extraction. Although this increase is modest compared to previous comparisons in tissues such as liver,6 note that bone marrow and cultured cells are among the best substrates for the organic extraction method in terms of high rates of packaging success. DNA yields were similar using both techniques. DNA purity was also indistinguishable, with A260/280 ratios of 1.8 among all samples (data not shown).

Conclusions

The list of tissues amenable to Stratagenes RecoverEase DNA isolation kit now includes blood, bone marrow, and cultured cell lines. With the kit, reduce the need for hands-on time, eliminate toxic organic solutions, and remove ethanol precipitation and lengthy rehydration steps. Other potential applications for genomic DNA recovery include Southern blot analysis, pulsed-field gel electrophoresis, PCR amplification, genomic library preparation, and shuttle vector recovery in transgenic in vivo mutation assays.

Methods8

Blood: Recover rodent whole blood (5 to 7 ml) with the anticoagulant ACD (0.48% citric acid w/v, 1.32% sodium citrate w/v, 1.47% glucose w/v),7 and add five volumes of Tris-buffered ammonium chloride (140 mM NH4Cl, 17 mM Tris-HCl, pH 7.65). After 5 minutes at 37C, pellet the lysed blood cells by centrifugation, then wash the pellet with NaCl (0.85%) solution.7 The pellet may be stored at 80C. When ready for use, thaw the pellet, and isolate and purify the DNA following the standard kit protocol.

Bone Marrow: Inject lysis buffer through two mouse femurs or one rat femur, and incubate the lysate on ice for 10 minutes; centrifuge and digest/dialyze per the standard kit protocol.

Cultured Cells: Suspend cells (1 to 5 x 106) in lysis buffer, eliminate the coarse disruption step, and follow the standard procedure.

REFERENCES
  1. Monforte, J.A., et al. (1995) Environ. Mol. Mutagen. 23 (Suppl. 23): 46.

  2. Rogers B.J., et al. (1996) Environ. Mol. Mutagen. 27 (Suppl. 27): 57.

  3. Short, J.M., et al. (1988) Fed. Proc. 8515a.

  4. Kohler, S.W., et al., (1991) Proc. Natl. Acad. Sci. USA 88: 7958-7962.

  5. Jakubczak, J.L., et al., (1996) Proc. Natl. Acad. Sci. USA 93: 9073-9078.

  6. Rogers, B.J., et al. (1996) Strategies 9 3: 73-74.

  7. Sambrook, J., et al. (1987) Molecular Cloning: A laboratory Manual,Second edition. Cold Spring Harbor Press. New York. p. 9.17.

  8. Rogers, B.J., (1998) Environ. Mol. Mutagen. 31 (Suppl. 29): 12.

* U.S. Patent Nos. 5,347,075 and 5,824,287 and 5,589,155; European Patent No. 0289121;Japanese Patent No. 2618973
** U.S. Patent No. 5,188,957


'"/>

Source:


Page: All 1 2 3 4 5 6 7 8

Related biology technology :

1. Eppendorf Multiporator Transfection Protocols for Eukaryotic Cells
2. Eppendorf Multiporator Transfection Protocols for Eukaryotic Cells
3. Optimizing DNA Amplification Protocols using the Eppendorf Mastercycler
4. High-Level Expression of Peanut Allergens Affected by Rare Codon Usage
5. Innovative Tissue Array Technology for High-Throughput Screening of Gene Expression
6. Highest Possible Transformation Efficiencies for High-Throughput Applications
7. High-Throughput Cultivation of Bacterial Clones
8. Safe Production of High-Titer Retrovirus
9. New Mammalian Expression Vectors Employ Stable, High-Level Fluorescence Humanized Renilla GFP Reporter
10. prostar RT-PCR Systems for Robust High-Fidelity RNA Amplification
11. pfuturbo DNA Polymerase: A High-Performance, High-Fidelity Enzyme Ideal for PCR Cloning
Post Your Comments:
*Name:
*Comment:
*Email:
TAG: New Protocols for Isolating High Molecular Weight Genomic DNA

(Date:6/1/2015)...  Turing Pharmaceuticals AG announced that life sciences executive ... MSc,  has joined the company as president of ... holds a medical degree as well as a ... accomplishments in the pharmaceutical industry, and extensive clinical ... played a leadership role in numerous programs for ...
(Date:6/1/2015)... and RARITAN, N.J. , ... LLC (Janssen) announced data from the Phase 3 ... in progression-free survival (PFS) with trabectedin (YONDELIS ® ... liposarcoma (LPS) or leiomyosarcoma (LMS) previously treated with ... regimen. SAR3007 is the largest randomized Phase 3 ...
(Date:6/1/2015)... 1, 2015 /PRNewswire/ - BIOREM Inc. (TSX-V: BRM) ("Biorem" or "the Company") ... 2015.  Biorem,s complete 2015 first quarter financial statements and MD&A have ... ended March 31,(in CDN$,000 except per share data) , ... , 1,974 Gross profit , 1,531 , ... (loss) , 528 , (531) Basic earnings (loss) ...
(Date:6/1/2015)... , June 1, 2015  The Val Skinner ... national innovative science curriculum for high school students focused ... 16 th annual LIFE Event (LPGA Pros in ... Ridge Country Club in West Caldwell, N.J. ... LPGA,s biggest stars raised more than $500,000 on Monday, ...
Breaking Biology Technology:Turing Pharmaceuticals Names Eliseo Salinas, MD and MSc, President of Research and Development 2Turing Pharmaceuticals Names Eliseo Salinas, MD and MSc, President of Research and Development 3Trabectedin Improves Disease Control in Patients with Advanced Soft Tissue Sarcoma 2Trabectedin Improves Disease Control in Patients with Advanced Soft Tissue Sarcoma 3Trabectedin Improves Disease Control in Patients with Advanced Soft Tissue Sarcoma 4Trabectedin Improves Disease Control in Patients with Advanced Soft Tissue Sarcoma 5Trabectedin Improves Disease Control in Patients with Advanced Soft Tissue Sarcoma 6Biorem Reports Increased Revenues and Earnings for First Quarter 2Biorem Reports Increased Revenues and Earnings for First Quarter 3Val Skinner Foundation Announces Partnership With Discovery Education 2
... England, April 21 Paul Capital,Healthcare and Vernalis ... a financing agreement relating to frovatriptan, Vernalis, product,for ... will receive,approximately euro 18.4 million in cash from ... will receive approximately 90 per cent,of Menarini,s payments ...
... MIT Lincoln Laboratory have developed a powerful sensor ... and smallpox in less than three minutes. , ... for THreatening Environmental Releases), represents a significant advance ... Lincoln Lab,s Biosensor and Molecular Technologies Group. Current ...
... b3c newswire /   -  DuoCort AB announced ... Optimized Care for Adrenal Insufficiency – at the 10th ... Congress Centrum in Berlin.  The symposium will be held ... delegates attending ECE 2008 are invited to the symposium. ...
Cached Biology Technology:Vernalis and Paul Capital Healthcare Sign euro 18.4 Million Financing Agreement 2Vernalis and Paul Capital Healthcare Sign euro 18.4 Million Financing Agreement 3MIT's PANTHER sensor quickly detects pathogens 2DuoCort Hosts Adrenal Insufficiency Symposium at the 10th European Congress of Endocrinology in Berlin 2
(Date:5/7/2015)... 7, 2015 Fingerprint Cards ... and FPC1035, FPC,s smallest touch fingerprint sensors to ... considered for integration on the backside of the ... increased possibilities to integrate touch fingerprint sensors in ... improves possibilities for module manufacturers to customize the ...
(Date:4/27/2015)... Apr. 27, 2015 Profile Solutions, Inc. (OTC: ... security systems is pleased to announce that Dr ... consultant and member of its scientific advisory board. ... as a thought leader in technology-enhanced learning models. He ... and has published studies and books focused on online ...
(Date:4/14/2015)... , April 14, 2015  HYPR Corp. ... Online (FIDO ® ) Alliance tm , an ... FIDO members commit to share technology and collaborate ... that are interoperable, more secure and private, and ... biometric identity verification that protects sensitive user information ...
Breaking Biology News(10 mins):FPC Introduces its Smallest Touch Fingerprint Sensors to Date 2Profile Solutions Names Medicinal Cannabis, Agriculture and Technology Expert Dr. Gerry Bedore to its Scientific Advisory Board 2Profile Solutions Names Medicinal Cannabis, Agriculture and Technology Expert Dr. Gerry Bedore to its Scientific Advisory Board 3Profile Solutions Names Medicinal Cannabis, Agriculture and Technology Expert Dr. Gerry Bedore to its Scientific Advisory Board 4HYPR Corp. Joins the FIDO Alliance 2HYPR Corp. Joins the FIDO Alliance 3
... the cells of blood vessels running through ovarian tumors. ... diagnosis and treatment for this disease. , The team ... Pennsylvania, and universities in Greece and Italy, used a ... ovarian tumors and four normal ovarian tissue samples. From ...
... markedly in their response to sleep deprivation, but the ... identify. Researchers have now found that a genetic difference ... particularly sensitive to the effects of sleep deprivation. The ... at the University of Surrey's Sleep Research Center, appear ...
... machine of their design, Duke University Medical Center researchers ... improve the ability of physicians to repair damaged joints ... experiments are successful, the scaffold could be used in ... Moutos, a graduate student in the Orthopedic Bioengineering Laboratory ...
Cached Biology News:Researchers identify ovarian cancer biomarkers 2Individual differences in a clock gene predict decline of performance during sleep deprivation 2Woven scaffolds could improve cartilage repair 2
... system is used for conventional PCR and ... MyCycler personal thermal cycler, which features a ... ml tubes or plates, programmable time, temperature, ... block modes of temperature control. The system ...
... Ciphergen's ProteinChip arrays have been ... from biological samples. A variety of ... of proteins according to their unique ... different spots, allowing side-by-side sample comparison, ...
The Freeze 'N Squeeze DNA gel extraction kit uses a quick freeze-and-thaw cycle and centrifugation to draw 50-23,000 bp DNA out of agarose gel slices. The kit includes materials for 100 preps....
The Econo gradient pump rack is a preassembled rack that is used to hold the components of the Econo gradient pump kits....
Biology Products: