Questions relating to protocols, procedures, and good practice when using laboratory equipment.

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3
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1answer
75 views

Good pipetting technique?

Good pipetting technique is essential for many biologists, but it can be hard to get right. When I take 1 µl of liquid using a micropipette, I seem to always take less than 1 µl, and that amount is ...
2
votes
0answers
12 views

How would one identify cellular transcription factors associated with a viral protein in a treated cell line?

I've been working as the computer guy for a microbiology lab for the past few months. I've always been interested in bench work, but my wet lab experience is rather limited and thus so is my ...
3
votes
1answer
19 views

Why is it advised to avoid bubble formation during mixing?

I have been told not to vortex solution containing protein. The reason I was given is bubble formation. Here I am interested in the effect of bubble formation in general.
1
vote
0answers
37 views

Making mistakes in the laboratory [closed]

I have just started an internship in a microbiology laboratory. I have had a rather small amount of laboratory experience before this so had to learn mostly everything from scratch. I have been there ...
0
votes
1answer
36 views

probe amplification in MLPA

I'm reading an article about MLPA (Multiplex ligation-dependent probe amplification) and I got stuck on this sentence: The advantage of splitting the probe into two parts is that only the ...
3
votes
1answer
42 views

Resolution of X-ray crystallography

A structure determined by X-ray crystallography has a resolution of 1.5 Å. When I look at the coordinates, I find every backbone C-N distance is 1.32 Å.i.e. Accurately predicted. If resolution is not ...
0
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0answers
17 views

What Helminth species has the simplest LABORATORY CONDITION for hatching?

I am very interested to hatch the eggs of a parasite in LABORATORY ! but for my first experience I need a species that is not so expensive or time consuming for experiment and can be done by an ...
3
votes
0answers
43 views

Why do I get such a strong background in a detection of DIG-labeled DNA (Southern Blot)?

I have done a Southern Blot Analysis of DNAmt transferred to nylon membranes. The DNA was firstly loaded on a 2% agarose gel. An immunoassay was done to detect the bands using Anti-Digoxigenin ...
1
vote
0answers
43 views

Why do PCR manufacturers recommend assembling the reaction mix on ice?

Many PCR manufacturers recommend preparing the reaction mix for the reaction on ice. For example, here is NEB's recommendation for their Q5 polymerase. Other manufacturers also include similar steps: ...
1
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0answers
16 views

Extraction of RNA from algae

I have used a standard protocol (I will give the bibliography below) to extract RNA from an algae (Posidonia) but I have get literally nothing, since I could not even see traces of the two rRNA. I ...
0
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0answers
10 views

Must one autoclave LB medium after it has been made? [closed]

Is it necessary to autoclave LB medium after it has been made?
3
votes
1answer
40 views

Is there a difference between Luria Broth and Lysogeny Broth?

Is there a difference between Luria Bertani and Luria Broth? Or are they both the same thing? Is it necessary to autoclave LB medium after it has been made?
0
votes
1answer
34 views

what benefits does lab procedure Realtime PCR have in gene silencing experiments? [closed]

RT-PCR performed in gene silencing but mechanistically what are the benefits of this procedure?
0
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0answers
23 views

My flow cytometry antibody won't reach saturation

I am staining human neutrophil populations (CD177) and then trying to get a different test target antibody to give a dose response to binding to this population. I have tried concentrations of the ...
0
votes
2answers
51 views

Getting PCR amplification at annealing higher than Tm!

I am amplifying a gene where in a gradient pcr i am getting amplification at an annealing temperature about 5 degrees (67) higher than Tm (62.5)? What is wrong here? Also, I am getting a very strong ...
4
votes
1answer
91 views

How sterile is working next to a bunsen burner?

When I was still doing lab work, many people would just wear gloves and work next to a bunsen burner because the clean benches were all in use. This was mostly for plating bacteria like Bacillus ...
3
votes
1answer
69 views

In CRISPR bacteria, how does viral genomes get integrated into the spacers of CRISPR? Also, in its use, where does Cas9 cut the DNA?

I've been out of Biology for about a year polishing my programming skills. I know CRISPR/Cas9 allows targeted 'cutting' of DNA via RNA-guidance. Few questions regarding this. Regarding to its ...
0
votes
0answers
12 views

Easiest way to digitally store pathlab reports,blood reports and other reports

I'm engineering student, and working on cheap way to store medical records of a person. I want to know way to partially digitize variety of reports, by partial I mean that I would store photo of ...
3
votes
0answers
24 views

How do I test if microbes have survived after dehydration?

I have a solution containing various bacteria and fungi. My aim is to place solution on filter paper, and wait until it dries. I then wish to test if the organisms have survived, either on the dried ...
1
vote
0answers
23 views

Batch several sequences for absent restriction sites

I have a collection of about 120 7kB sequences I would like to check for ether a list of specific restriction sites, or what restriction sites might be absent in all of them. Is there a app or ...
0
votes
1answer
49 views

On which amino-acids residue is the SDS acting on?

I would like to know exactly what is the mechanisme of the SDS, and I would like to know on which amino-acids residue the SDS is acting on. Can you help me please ? Thank you in advance !
0
votes
0answers
16 views

Quantitative measurement of taxis

Is there a way to quantitatively measure different forms of taxis, like chemotaxis, electrotaxis, phototaxis, etc. Along with that, how can we also quantitatively compare the strengths of the ...
2
votes
1answer
39 views

Where does the inverse seconds unit come from in the association constant?

I'm working to determine Kd(s) kinetically by generating association and disassociation curves. Kon (association constant, or on-rate) is in inverse seconds multiplied by inverse molar. I get that ...
1
vote
1answer
40 views

Making positive charged polyacrylamide

I am interested in positive charged polyacrylamide to electrophorese molecules I am interested in. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2643323/ ...
2
votes
3answers
227 views

Amplification technique for proteins similar to PCR for DNA?

I know PCR can be used to amplify a tiny sample of DNA in order to perform experiments. Is there a similar technique to use on a protein sample? More specifically, I'm not interested in "cutting" up ...
3
votes
1answer
63 views

How C. Elegans is used for siliencing genes

The experiment that is using C. Elegans to silence the Genes. I have a question about Why and how C. Elegans can use the DNA plasmid that is generated with the gene of interest in the bacteria by ...
1
vote
2answers
90 views

Extending a small fragment of DNA

Is there a way to extend a small fragment of DNA, say 150 bp, by making copies of itself and attaching each copy of that small fragment to the end of that 150 bp sequence? For example, I want a ...
2
votes
3answers
117 views

Is RNase AWAY in the lab dangerous?

I use RNase AWAY in the lab. I would like to know how dangerous this chemical is for health. For example, when I remove my gloves my hands smell because of the RNAse AWAY
2
votes
0answers
162 views

How much salt [NaCl] is too much in DNA precipitation?

In DNA extractions, how much is too much salt in a CTAB extraction buffer? Protocols hover around 2.5 molar; if you go over this (e.g. 25 molar), will you saturate your solution, and precipitate the ...
2
votes
2answers
59 views

Career progression through biosafety levels?

Does a career in infectious disease typically progress through biosafety levels, or do people select one and specialize their training in procedures specific to those hazards? That is, say Dr. ...
4
votes
1answer
121 views

When did mouth pipetting stop becoming a way to handle liquids in a lab?

Almost all modern lab protocols have an addendum prohibiting pipetting by mouth, instead mandating that a Gilson pipette, a rubber pipette ball, or a serological Pipet-Aid be used. However, it was ...
4
votes
2answers
164 views

How to Extract RNA and perform RT-qPCR from very few cells ( ~5,000)?

I am currently conducting an experiment which involves FACS-sorting a specific population of cells using an antibody of interest. In order to validate the type of cells I have collected using this ...
1
vote
0answers
109 views

Interpretation of qPCR results for low expression genes

I am attempting to validate existence of a transcript using 40 cycle qPCR. I designed primers for a unique feature of this transcript, and also designed primers for a sequence in the transcript that ...
4
votes
2answers
68 views

Why lab technicians use indirect (antibody reaction) method for diagnosing?

In microbiology we have two types of microbial diagnosis. The direct method is where we detect the invader's DNA, Antigens or culture to see the exact pathogen while the second, indirect, method is ...
2
votes
1answer
77 views

Why does my anti-ubiquitin antibody visualization not work on my PAGE gel?

I am using 2D gel electrophoresis to visualize polyubiquitinated proteins. However, while I can see actin and heat shock protein using when appropriate antibodies, I cannot visualize them using ...
2
votes
2answers
136 views

Why do many DNA solutions contain additional compounds?

DNA solubility data in only water is scarce. A previous question asked for a quantification of DNA solubility in water. It seemed like it would be easily answerable, however isn't quite that simple ...
2
votes
3answers
123 views

LCMS/MS versus Western Blot

I have a general question regarding which method would you recommend me to use if I would like to investigate the difference in the level of several proteins in tissue samples and compare different ...
1
vote
3answers
202 views

Trypsin and cell culture

I am doing an experiment where I have treat the cells with a drug and calculate their counts. I would like to know if is bad to trypsinize the cells in consecutive days i.e. twice within 48 hours. How ...
5
votes
1answer
41 views

One Sample, Different Methylation Values

I'm hoping that somebody here can explain this unusual result that I'm getting with some pyrosequencing data. I have bisulphite converted a few samples a couple of times over the years for different ...
5
votes
1answer
120 views

Is there a protocol for freezing and thawing Bacillus subtilis cells?

There is a book that says to store Bacillus spores in 50% glycerol at -70 degrees Celsius (doesn't mention if the 50% is final concentration or not). But from what I know, the cells themselves can be ...
12
votes
2answers
641 views

How sterile is sterile when working with nucleic acids to prevent contamination?

I am reading up on preparatory work on working with nucleic acids and a lot of the instructions speak on excessive procedures on cleaning environments with high %ethanol and making sure the equipment ...
2
votes
1answer
29 views

Do I have to use sucrose to induce a lac promoter?

I'd like to optimize the expression of a Fab fragment in Escherichia coli. For induction of the lac promoter on the pAK400 vector I use IPTG and sucrose. Do I optimize the expression in case I would ...
1
vote
0answers
109 views

Recommend any Molecular lab LIMS (Laboratory Information Management System)

We are looking to develop or customize a LIMS for our molecular lab. Do you know of a LIMS that you've used in a molecular lab before, or one that could be used. Thanks for the help (Edit) Some of ...
6
votes
1answer
310 views

Extracting cell-free DNA

Has anybody had any success in extracting cell free DNA from plasma without using expensive kits? I've already spent a lot of my own blood trying to use standard phenol/chloroform methods with very ...
7
votes
1answer
805 views

Magnetic-activated cell sorting vs. FACS

When sorting cell populations it is possible to use either magnetic-activated cell sorting or fluorescence-activated cell-sorting (FACS). I am wondering when you would choose either technique and what ...
3
votes
1answer
41 views

Decreasing Solubility as Solvent Moves Higher in Paper Chromatography

In paper chromatography, as the mobile phase (I used 1:9 acetone/petroleum ether) climbs higher and higher, will the solubility of the solutes decrease as the solvent moves higher? I speak mostly ...
2
votes
1answer
36 views

Workspace preparation and cleanup for DNA work [duplicate]

What steps should be taken in a molecular lab environment to help ensure that DNA samples/stocks are not contaminated, or contaminate other objects in the lab?
2
votes
2answers
452 views

Using an analytical balance (0.0000 precision) to measure µg (micrograms)?

If i needed to prepare 200µg/ml of proteinase K, and the proteinase K was in a solid powder form, would I have to weight out 200 µg using an analytical balance, and if so, is it possible with a ...
-1
votes
1answer
946 views

How to make µg/ml concentrations of proteinase-K?

How does one prepare concentrations in the mass/volume (weight/volume) form, for substances like nucleic acids or in this case, proteinase? A detailed example would be helpful. I need to prepare ...
9
votes
1answer
363 views

RNA migrating slower than DNA on Formaldehyde Gel?

So I ran into an interesting problem. I'm getting a linear DNA band that is twice as long (4x bases, but as denatured probably only 2x) as an RNA band running at the same size in a formaldehyde gel. ...