Navigation Links
High-Throughput Cultivation of Bacterial Clones


Culture 192 clones simultaneously with the tempest thermoshaker

Jeff Braman
Stratagene

The tempest thermoshaker and a 96-deep-well round-bottom plate were used to cultivate bacterial clones in a high-throughput format. Early- and mid-log phase rates of bacterial growth were equivalent in aerated and nonaerated wells of the plate. Late-log phase growth rates of bacteria cultured in nonaerated wells were only slightly slower than growth rates measured in aerated wells. Final cell densities in aerated and nonaerated wells were identical. Flask-cultured bacteria demonstrated a slower initial rate of growth compared to plate-cultured bacteria, however, mid-log phase growth and final cell density were equivalent to plate-cultured bacteria. Well-to-well plate contamination was undetectable. Equal quantities of plasmid DNA were recovered from equal volumes of plate- and flask-cultured bacteria sampled at the end of the incubation period.

The Tempest thermoshaker is a compact benchtop incubator and shaker that is used for regulated temperature incubation (0 to 40C) and rotary mixing (200 to 1500 rpm) of multiple samples. The unit is supplied with a holder to accommodate fifty six 1.5-ml tubes. With Stratagenes minitube adapters, the thermoshaker can also hold 0.5- or 0.6-ml tubes. A microplate platform, also available, holds four, 96-well microtiter plates or two, 96-deep-well plates. The latter plates are particularly useful for cultivating bacteria in a high-throughput.

96-Well Plate vs. Flask Cultivation of Bacteria

Growth rates and final cell densities were measured for plate- and flask-cultured Epicurian Coli XL1-Blue bacteria containing the pBluescript plasmid. One ml of LB medium, containing 100 mg/ml ampicillin, was added to each 2-ml well of the 96-well plate. Bacteria were inoculated into alternate wells of the plate at a density of 2 105 cells/ml. The plate was sealed with its flexible plastic lid, which is designed to seal each well to prevent cross contamination. Perforations were made in the lid over the first 48 wells to allow an exchange of gases between the air space above the culture medium and the atmosphere. The 96-deep-well plate was then incubated at 37C in the thermoshaker (600 rpm).

Bacteria at the same cell density were inoculated into a 2-liter flask, which held 1 liter of LB medium containing 100 g/ml ampicillin. The flask was covered with a piece of sterile aluminum foil and incubated in a New Brunswick Scientific Model G25 Series floor incubator-shaker at 300 rpm.

Bacterial growth was monitored at various time points by removing samples from the aerated and nonaerated wells of both the plate and flask, then measuring absorption at 550 nm.1 Bacteria cultured in nonaerated wells of the plate were removed by perforating the plate lid with a hot needle just prior to sampling. This method prevented the need to remove the lid from the plate and introduce air into the nonaerated wells of the plate.

Bacterial Growth Rates

Figure 2 demonstrates equivalent growth rates of bacteria in early- and mid-log phases in aerated and nonaerated wells of the plate. Late-log phase growth rates of bacteria cultured in nonaerated wells were only slightly slower than in aerated wells. Cell densities at the end of the incubation period in aerated and nonaerated wells were identical. Flask-cultured bacteria demonstrated a slower initial rate of growth compared to plate-cultured bacteria; however, mid-log phase growth and final cell density were equivalent to plate-cultured bacteria. No 550-nm absorption above background was observed in noninoculated wells of the plate.

A sample from every well of the plate was streaked onto an LB-ampicillin agar plate with a sterile toothpick and the plate was incubated at 37C for 24 hours. Bacterial growth was observed only from wells initially inoculated with bacteria. Bacterial growth was not observed in samples retrieved from noninoculated wells (data not shown). Therefore, cross contamination to alternate noninoculated wells did not occur in the aerated and nonaerated wells of the plate.

Plasmid Yield from Plate- and Flask-Cultured Bacteria

Plasmid DNA was isolated from 1 ml of plate- (aerated and nonaerated wells) and flask-cultured bacteria collected at the end of the incubation time period using the StrataPrep plasmid miniprep kit. Plasmid DNA yields were equivalent in plate- and flask-cultured bacteria, as judged by gel electrophoresis (Figure 3).

Fig.3

Conclusions

The Tempest thermoshaker allows 192 bacterial clones to be cultivated simultaneously in two, 96-deep-well plates. A special lid seals each well and prevents cross contamination. Bacterial growth rate, final cell density, and plasmid yield are virtually identical in aerated and nonaerated plate- and flask-cultured bacteria. The Tempest thermoshaker is a convenient and valuable tool when high-throughput capability is required.

REFERENCES
  1. Miller, J.H. (1972) Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. pp. 32-33.


'"/>

Source:


Page: All 1 2 3

Related biology technology :

1. Innovative Tissue Array Technology for High-Throughput Screening of Gene Expression
2. Highest Possible Transformation Efficiencies for High-Throughput Applications
3. High-Throughput Isolation of Total RNA
4. High-Throughput System Generates Ultra-Pure PCR Products Suitable for All Applications
5. The DIG System Nonradioactive Automated High-Throughput In Situ Hybridization: a Powerful Tool for Functional Genomics Research
6. Advantages of ReadyAgarose Precast Gels Over 96-Well Handcast Gels for High-Throughput Analysis, Rev A
7. Mammalian and Bacterial Expression in One Vector
8. Bacterial mRNA Isolation, Fast and Easy
9. Bacterial RNA Isolation From Infected Eukaryotic Hosts
10. Bacterial RNA Isolation From Infected Eukaryotic Hosts
11. Bacterial Whole Genome Array Analysis
Post Your Comments:
*Name:
*Comment:
*Email:
TAG: High Throughput Cultivation Bacterial Clones

(Date:3/4/2015)... (PRWEB) March 04, 2015 Follow ... represents one of the fastest growing technologies in the ... in subcellular visualization is driving growth in nucleic acid ... deeper understanding of gene expressions will continue to benefit ... applications in fields ranging from pharma, biotech, food & ...
(Date:3/4/2015)... , March 4, 2015 ... Paris en décembre 2011, ... SARAH   », qui compare   l ... le   sorafénib dans le carcinome hépatocellulaire avancé, ... cancer du foie avancé   ; les résultats ...
(Date:3/3/2015)... SAN DIEGO and TORONTO, March 3, 2015 /PRNewswire/ - ... company developing new therapeutics and molecular diagnostics that target ... for the quarter and seventh-month period ended December 31, ... changed its fiscal year end from May 31 to ... periods we are reporting today are for the quarter ...
(Date:3/3/2015)... March 3, 2015 NASA astronaut Scott Kelly ... month to spend a year living and working on the ... from 5:30 to 7 a.m. EDT Monday, March 9. ... as he completes the final weeks of his training. The ... on NASA Television highlighting his mission training and previous spaceflights. ...
Breaking Biology Technology:Focus on Quantifying Nucleic Acid Molecules Spurs Growth in the qPCR & dPCR Instrumentation Market, According to a New Report by Global Industry Analysts, Inc. 2Focus on Quantifying Nucleic Acid Molecules Spurs Growth in the qPCR & dPCR Instrumentation Market, According to a New Report by Global Industry Analysts, Inc. 3Focus on Quantifying Nucleic Acid Molecules Spurs Growth in the qPCR & dPCR Instrumentation Market, According to a New Report by Global Industry Analysts, Inc. 4Traitement du cancer primitif du foie : Recrutement terminé pour l'étude SARAH et résultats attendus fin 2016 2Traitement du cancer primitif du foie : Recrutement terminé pour l'étude SARAH et résultats attendus fin 2016 3Traitement du cancer primitif du foie : Recrutement terminé pour l'étude SARAH et résultats attendus fin 2016 4Traitement du cancer primitif du foie : Recrutement terminé pour l'étude SARAH et résultats attendus fin 2016 5Traitement du cancer primitif du foie : Recrutement terminé pour l'étude SARAH et résultats attendus fin 2016 6Aptose Biosciences reports results for the quarter and seven months ended December 31, 2014 2Aptose Biosciences reports results for the quarter and seven months ended December 31, 2014 3Aptose Biosciences reports results for the quarter and seven months ended December 31, 2014 4Aptose Biosciences reports results for the quarter and seven months ended December 31, 2014 5Aptose Biosciences reports results for the quarter and seven months ended December 31, 2014 6Aptose Biosciences reports results for the quarter and seven months ended December 31, 2014 7Aptose Biosciences reports results for the quarter and seven months ended December 31, 2014 8Aptose Biosciences reports results for the quarter and seven months ended December 31, 2014 9Aptose Biosciences reports results for the quarter and seven months ended December 31, 2014 10Aptose Biosciences reports results for the quarter and seven months ended December 31, 2014 11Aptose Biosciences reports results for the quarter and seven months ended December 31, 2014 12NASA Astronaut Scott Kelly Available for Interviews before One-Year Space Station Mission 2
... are you? How about your firm? Slow? Fast? Lightning Speed? Not ... because you have yet to adopt a wiki. A wiki, you ... quick or fast. Today, it has come to mean a collaborative ... armamentarium of Web 2.0 that encourage conversations and draw on the ...
... move designed to give Wisconsin a competitive advantage over ... agreement with the Wisconsin Alumni Research Foundation ... non-profit and academic institutions in Wisconsin a free, non-exclusive ... WARF. , ,In what the administration called a landmark ...
... - A defining moment in the first televised debate ... 67-year-old man from Brown Deer, asked Gov. Jim Doyle ... help turn around Milwaukee's economy. Beene, who had worked for ... perception that manufacturing plants "are closing all over the place" ...
Cached Biology Technology:Joining the wiki wacki world 2Joining the wiki wacki world 3Joining the wiki wacki world 4Joining the wiki wacki world 5Doyle, WARF announce partnership to lure stem cell companies 2Doyle, WARF announce partnership to lure stem cell companies 3Candidates' sparring over jobs focuses debate on right issue 2Candidates' sparring over jobs focuses debate on right issue 3
(Date:2/5/2015)... 2015  It is gratifying to see that the ... genomic science as a means to better understand human ... I was honored to participate in today,s White House ... Since the 1980s my teams have ... first sequenced genome of a free living organism, the ...
(Date:1/22/2015)... , Jan. 22, 2015 Research and Markets ( ... - A Global Market Overview" report to their offering. ... is to ensure that an individual is who she/he is ... make use of a person,s unique physical characteristics, such as ...
(Date:1/22/2015)... , Jan. 13, 2015 /PRNewswire/ - Today, FindBiometrics, the leading source ... launch of its new website design. "When we ... infancy", says Peter O,Neill , founder and CEO of ... industry needs involvement from the key players on a very ...
Breaking Biology News(10 mins):J. Craig Venter, Ph.D., Co-Founder and CEO, Human Longevity, Inc. (HLI) Participates in White House Precision Medicine Event 2Biometrics - A Global Market Overview 2Biometrics - A Global Market Overview 3Identity Industry Leading Publication Debuts New Look As Biometrics Hit Mainstream 2
... CINCINNATIThe American Cancer Society estimates one man in six will ... cause of cancer death in men. With a new partnership ... a new drug in stopping the growth of prostate cancer ... of Zhongyun Dong, PhD, will test the efficacy of a ...
... Johns Hopkins University School of Medicine have been awarded ... institutions. Andrew P. Feinberg, M.D., M.P.H., King Fahd ... for Epigenetics, will receive the honorary doctorate from the ... Stockholm City Hall on May 7. Craig Montell, ...
... China are reporting that they have found a way to ... that have been reprogrammed into a primordial, embryonic-like state are ... published online last week by the Journal of Biological ... Academy of Sciences point to a marker they found in ...
Cached Biology News:UC to test targeted treatment for prostate cancer 22 Hopkins scientists awarded European honorary doctorates 2Chinese scientists discover marker indicating the developmental potential of stem cells 2
Prepared in distilled water....
... Origami host strains are K-12 derivatives ... reductase (trxB) and glutathione reductase (gor) genes, ... the cytoplasm. Studies have shown that expression ... than in another host even though overall ...
Donor Horse Serum US Origin...
Mouse Aminopeptidase P2/XPNPEP2 Biotinylated Aff Pur PAb Keywords: AminopeptidaseP2, Metalloproteinase, Metalloprotease, X-Prolyl Aminopeptidase 2, X-Pro Aminopeptidase 2, Membrane-bound Prote...
Biology Products: