Microbial Molecular Biology

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3 How much amount (in ng) of vector or insert required for restriction or ligation reaction?
usually i used 20 microliters restriction reaction contain 30-100 ng vector or insert. Its correct or i can change my concentration and also i used 1:... [more]

usually i used 20 microliters restriction reaction contain 30-100 ng vector or insert. Its correct or i can change my concentration and also i used 1:3 ratio (Vector:Insert) for ligation reaction. Till now i didnt get single colony. Please help me. Restriction Setup for Double Digest: insert - 0.5 -7 Microliters ( 35 ng/microliters) R.Enzyme I - 1 Microliter R. Enzyme II - 1 Microliter NEB Buffer - 2 Microliter BSA - 0.5 Microliter NF. Water - 8.5 - 15 microliters Total Reaction - 20 Microliters 2 hours@37 C Reaction arrest 65 C @ 20 minutes Ligation Setup Vector - 5 Microliters Insert - 15 microliters Ligase enzyme- 1 Microliter Ligase Buffer- 2 Microliters NF.Water - 2 Microliters Total Reaction - 25 Microliters Overnight (1-16 Hours) Reaction Arrest 65 C @ 20 Minutes Transformation Using DH5alpha ( heat shock - 42 c for 2 Min and cold shock- ice cubes for 2min) 1- 2 hour shaking @ 37 c Pour Plate Method to spread my culture(100 Microliters) into agar plate (Amp-0.1 milligram/ml) Next day - No colonies

By Senthil Kumar Sankareswaran · Anna University, Chennai

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Question:
4 Is DNA hydrophilic or hydrophobic?
It is mentioned in the paper that "The presence of eDNA created a highly hydrophobic bacterial cell surface", which quite confused me. DNA is a hydrop... [more]

It is mentioned in the paper that "The presence of eDNA created a highly hydrophobic bacterial cell surface", which quite confused me. DNA is a hydrophilic macromolecule, how come it makes the surface hydrophobic?

Role of extracellular DNA in initial bacterial adhesion and surface aggregation. × Theerthankar Das, Prashant K Sharma, Henk J Busscher, Henny C van der Mei, Bastiaan P Krom

By Guanghong Zeng · Aarhus University

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Question:
4 Company where 1.6kb nucleotide sequence can be ordered to synthesize?
Where can we order to synthesize a 1.6kb long nucleotide sequence? We have the complete sequence ready with us. Can it be done where the normal primer... [more]

Where can we order to synthesize a 1.6kb long nucleotide sequence? We have the complete sequence ready with us. Can it be done where the normal primer synthesis is done?

By Santosh Keisam · Institute of Bioresources and Sustainable Development

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Question:
New Are gram-negative microbes better communicators than gram-positive ones vis a vis the paper here?
Periplasm may be an advantage to gram-negative microbes over gram-positive ones.

Periplasm may be an advantage to gram-negative microbes over gram-positive ones.

Exocytosis from gram-negative bacteria for Salmonella invasion of chicken ileal epithelium. × R C YashRoy

By Rakesh Yashroy · Indian Veterinary Research Institute

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Question:
11 What is a successful method for performing site-directed mutagenesis?
I have a couple of unwanted cut sites on a plasmid (roughly 5kb) that I would like to get rid of. Has anyone had success with a commercial kit or othe... [more]

I have a couple of unwanted cut sites on a plasmid (roughly 5kb) that I would like to get rid of. Has anyone had success with a commercial kit or other methodology in a similar situation?

By Swati Sureka · Cornell University

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Question:
1 Adsorption of DNA makes bacterial cells hydrophobic?
I have seen results from surface contact angle measurements that quite confused me. They described a reduced contact angle after removal of DNA from b... [more]

I have seen results from surface contact angle measurements that quite confused me. They described a reduced contact angle after removal of DNA from bacteria with DNase. As far as I know, DNA is a hydrophilic biomacromolecule. How come the bacterial surface becomes less hydrophilic after DNA is removed? Das, T., P. K. Sharma, H. J. Busscher, H. C. van der Mei and B. P. Krom (2010). "Role of Extracellular DNA in Initial Bacterial Adhesion and Surface Aggregation." Applied and Environmental Microbiology 76(10): 3405-3408.

By Guanghong Zeng · Aarhus University

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Question:
53 Can anyone suggest companies that sell TA cloning vectors?
I don't want the entire kit for cloning, instead I wish to buy only the vector i.e. TA cloning vector. Does anyone have any suggestions?

I don't want the entire kit for cloning, instead I wish to buy only the vector i.e. TA cloning vector. Does anyone have any suggestions?

By Kashif shamim · Goa University

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Question:
1 How to determine the copy number of a gene if the genome is unknown?
I am interested in a synthesis gene of quorum sensing molecules in a bacteria whose genome is unknown.

I am interested in a synthesis gene of quorum sensing molecules in a bacteria whose genome is unknown.

By Daniel Deng · University of Southern Mississippi

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Question:
6 What possible factor/molecular mechanisms help the amastigote form of Leishmania to remain undetectable inside the macrophage?
One aspect of the Leishmania parasite’s virulence is its ability to change forms from the flagellated promastigote form into the unflagellated ovoid... [more]

One aspect of the Leishmania parasite’s virulence is its ability to change forms from the flagellated promastigote form into the unflagellated ovoid amastigote form. As an amastigote, the parasite is ingested by the host’s macrophages. Inside the macrophage, the amastigote remains unaffected by the host’s immune response and able to multiply until the cell lyses, releasing the amastigotes.

By Tija Saha · Friedrich-Schiller-University Jena

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Question:
2 How much DNA yield can I expect following the DNA protocol mentioned in the article?
I need to extract DNA from puccinia striiformis stripe rust spores.

I need to extract DNA from puccinia striiformis stripe rust spores.

Whole genome amplification of the rust Puccinia striiformis f. sp. tritici from single spores. × Yanchun Wang, Mingqi Zhu, Rong Zhang, Hanli Yang, Yang Wang, Guangyu Sun, Shelin Jin, Tom Hsiang

By Raju Yadav · Sandor Proteomics

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Open Can anyone tell me what is the free phosphate concentration inside eukaryotic cells, especially in macrophages?
...

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By Somya Aggarwal · Jawaharlal Nehru University

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Question:
2 Possibility of two bacteria belonging to the same genus and species but having a high variation in hydrophobicity?
Both the strains are from Bacillus amyloliquefaciens and have very high difference in the hydrophobic nature of the cell surface. It was calculated us... [more]

Both the strains are from Bacillus amyloliquefaciens and have very high difference in the hydrophobic nature of the cell surface. It was calculated using Hexa decane as organic phage.

By Santosh Kumar · Bharath University

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Question:
35 How can I measure the amount of cells in a solution with filamentous fungi?
I would like to know how much fungi I add to my samples. I ususally grow them in liquid culture and use a mixer to try to homogenize the solution, the... [more]

I would like to know how much fungi I add to my samples. I ususally grow them in liquid culture and use a mixer to try to homogenize the solution, then I add a certain amount to each sample. But it seems that the amount of fungi added to each sample was very different. For the next time I would therefore like to know how much I add. Would it be useful to measure OD for example, even though the solution is not completely homogenized? Or has anyone used any other method that might work here?

By Rebecka Ringman · SP Technical Research Institute of Sweden

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Question:
1 Can anyone suggest a way to reverse transcribe bacterial RNA for real time gene expression studies?
If random hexamers are to be used please suggest which company we should use.

If random hexamers are to be used please suggest which company we should use.

By Jaya Chakraborty · National Institute of Technology Rourkela

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Question:
2 Checking coverage and specificity for yeast specific primer?
Is there any database or software where I can check the specificity of a yeast specific primer which targets 26S rRNA? For 16S rRNA of bacteria, we no... [more]

Is there any database or software where I can check the specificity of a yeast specific primer which targets 26S rRNA? For 16S rRNA of bacteria, we normally used RDP or SILVA for this purpose. But, I have no idea for yeast. Any suggestions and insights will be really helpful.

By Santosh Keisam · Institute of Bioresources and Sustainable Development

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Question:
6 How can I extract DNA from a Halophilic strain specifically Halomicrobium mukohataei?
I have tried many procedures but no results, I am successful in extracting a pellet but without DNA.

I have tried many procedures but no results, I am successful in extracting a pellet but without DNA.

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Question:
9 Does anyone have a good protocol for DNA extraction from deep sea sediment?
I have been following the method "SDS-based DNA extraction method" of Zhou et al. 1993. DNA Recovery from Soils of Diverse Composition. AEM. but not g... [more]

I have been following the method "SDS-based DNA extraction method" of Zhou et al. 1993. DNA Recovery from Soils of Diverse Composition. AEM. but not getting 16S rDNA amplification from universal primer (27F and 1492R). Primers are working well.

By Arvind Kumar · University of North Bengal

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Question:
4 How would you ensure house keeping genes in RT-PCR will not be affected when bacteria are exposed to certain stressors?
We plan to monitor the expression of certain virulence genes during acid shock treatment, We would like to know if the housekeeping genes used will al... [more]

We plan to monitor the expression of certain virulence genes during acid shock treatment, We would like to know if the housekeeping genes used will also be affetcted by this exposure?

By Atheesha Ganesh · University of KwaZulu-Natal

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Question:
10 What do you think about shipping bacteria on membrane filter a long distance (Africa to Asia)? How to avoid contamination?
I would like to ship water filtrates for further DNA extraction, but I'm afraid of contamination.

I would like to ship water filtrates for further DNA extraction, but I'm afraid of contamination.

By Edwige Tiodjio · Toyama University

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Question:
2 The best protocol for anaerobic bacterial DNA isolation(both Gm +ve &Gm -ve).
I want to do isolation DNA from anaerobic bacteria so i need a suitable protocol.

I want to do isolation DNA from anaerobic bacteria so i need a suitable protocol.

By Dolat Shekhawat · Indian Institute of Technology Rajasthan

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Question:
7 I am cloning a 4400 base pair gene using pET-28a vector. After many attempts I am not successful. Please suggest how can I get a full gene.
I have also done it in two parts. For one part I got the clone, for part 2 I am not getting any result. I have also changed temperature parameters .

I have also done it in two parts. For one part I got the clone, for part 2 I am not getting any result. I have also changed temperature parameters .

By Prashant Sharma · Panacea Biotec Ltd.

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Question:
Open Is there anyone who works with Gram positive and Gram negative bacteria transformed with Green Fluorescent Protein (GFP)?
I'm developing a method for DNA extraction from soil, and these transformed bacteria would be great to use as a standard for DNA/cell quantification. ... [more]

I'm developing a method for DNA extraction from soil, and these transformed bacteria would be great to use as a standard for DNA/cell quantification. Could anyone send me a sample of these or put me in contact with someone who is actually working on that? It would be a lot of help and could be a great collaborative work.

By Daniel Kumazawa Morais · Universidade Federal de Viçosa (UFV)

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Question:
7 SalI and XhoI ligation problem
I am doing ligation: Vector (6.3Kb) is SalI digested, digestion reaction checked on gel, reaction inactivated by heat, CIP treated, purified by kit, ... [more]

I am doing ligation: Vector (6.3Kb) is SalI digested, digestion reaction checked on gel, reaction inactivated by heat, CIP treated, purified by kit, checked on gel for quantity and quality. Insert (3kb) XhoI digested, digestion reaction checked on gel, reaction inactivated by heat, gel purified by kit, checked on gel for quantity and quality. Tried using CIP treatment for insert and without CIP treatment. Ligation reaction molar ratio vector: insert used = 1:3 or 1:6 used Ligation using T4 ligase Ligation using Takara ligation mixture My main problem is I am getting only my vector on my ligation mixture plates. Any suggestion or tips for my problem would be appreciated.

By Imran Khan · University of Incheon

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Question:
1 Does anyone offer training on bacterial taxonomy.
Kindly let me know if there so. I must go for a hands on training to work in actinobacterial taxonomy.

Kindly let me know if there so. I must go for a hands on training to work in actinobacterial taxonomy.

By Sivasankar Palaniappan · Annamalai University

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Question:
2 How can I design and select the best primer for qRT-PCR of genes of a Cyanobacteria (Prokaryotic cell)?
I would like to design and select primers for qRT-PCR of genes from Cyanobacteria which is Prokaryotic organism. I need to know which parameters are c... [more]

I would like to design and select primers for qRT-PCR of genes from Cyanobacteria which is Prokaryotic organism. I need to know which parameters are crucial to select the best primers. I would like to use SYBR Green method for PCR running. It would be highly appreciated if someone give me some suggestions detailing the procedure of selecting that primers.

By Khan Rezaul · Shanghai Jiao Tong University

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Question:
Open What is the role of tryptophanase in E.coli?
If adding cysteine or serine into medium, will the strain transform them into trytophana, and then further into indole?

If adding cysteine or serine into medium, will the strain transform them into trytophana, and then further into indole?

By Qiao Ma · Dalian University of Technology

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Question:
6 Problem with pCVD442 plasmid transformation - no colonies, eventhough competent cells are good.
I am doing a simple chemical method transformation using freshly made competent E.coli MC1061 cells. My competent cells are fresh and I checked the ef... [more]

I am doing a simple chemical method transformation using freshly made competent E.coli MC1061 cells. My competent cells are fresh and I checked the efficiency using another vector, works perfectly. I am using Ampicillin resistance marker 100 micro-gram/ml. As far as I know, I am taking care of ampicillin stock, by filtering and wrapping with aluminium foil. I am scrutinizing every single step but I am not finding the exact answer. After transformation there are no colonies. Is there anybody who has suggestions for me?

By Imran Khan · University of Incheon

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Question:
Open How does the bacterial cytoskeleton influence cell wall form?
Very recently, these proteins discovered as a framework of rod and spiral-shaped bacteria.

Very recently, these proteins discovered as a framework of rod and spiral-shaped bacteria.

By Roozbeh Yalfani · Islamic Azad University

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Question:
1 Does anybody knows the origin of putative phage recombination protein?
A RecT famlily protein is defined to be a putative phage recombination protein. We find that the putative phage recombination proteins encoded by both... [more]

A RecT famlily protein is defined to be a putative phage recombination protein. We find that the putative phage recombination proteins encoded by both bacteria and phage. What's the function of these phage recombination proteins and what's the origin of these bacteria genes? Do they come from phage infection or not?

By Yajie Yin · Chinese Academy of Sciences

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