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

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3
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1answer
50 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 ...
13
votes
1answer
6k views

When running gels what is the difference between constant volts or constant amps?

In general, you want to be consistent with running your gels either at constant volts or constant amps. However, it is very clear that during the progression of both PAGE and agarose gels, the free ...
0
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0answers
22 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
57 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 ...
3
votes
1answer
148 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 ...
1
vote
0answers
92 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
vote
1answer
135 views

Can you measure plasma glucose with a regular glucose meter?

I'm wondering if plasma glucose can be measured with a regular glucose meter and strips like this. I know these meters are normally used to measure whole blood glucose, but can they measure plasma ...
0
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1answer
44 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?
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0answers
23 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 ...
3
votes
1answer
97 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?
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0answers
11 views
4
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1answer
348 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 ...
2
votes
1answer
255 views

How long can a human neuron live outside the body in a controlled environment?

Have there been any experiments that have kept neurons alive (stationary), without preserving methods such as freezing? If yes, then how long were the cells kept alive for?
0
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0answers
13 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
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0answers
27 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
28 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 ...
1
vote
1answer
46 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/ http://onlinelibrary.wiley.com/doi/10.1002/bip....
0
votes
1answer
65 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 !
11
votes
3answers
642 views

How can I label cryotubes in a way that eliminates the problem of legible hand-writing?

My lab stores biological material (tissue, cells, plasma, serum) in a -130 C, liquid nitrogen freezer. The cryotubes that we use to store a samples are labelled by hand which frequently creates ...
7
votes
2answers
419 views

What is the best way to express two proteins in a mammalian cell?

I have two proteins and I will be preparing a vector with both genes for stable transfection. Each protein will have their own promoter and I will use piggyBac vector to insert a single cassette with ...
0
votes
0answers
18 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
54 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 ...
3
votes
1answer
81 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 ...
5
votes
1answer
141 views

Normal ECG/EKG Measurement?

I'm getting started using ECG using a 3 lead system by iWorx. I have place the leads on my two wrists and the ground on my ankle and have recorded some data into the provided LabScribe 3 software ...
2
votes
3answers
286 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
3
votes
0answers
310 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 ...
9
votes
1answer
538 views

Why are cell lines frozen in vapor phase?

For most of the cell lines I've come across, ATCC recommends storing them in the vapor phase of liquid nitrogen. I'm taking that to mean any place above the level of liquid in the nitrogen storage ...
4
votes
2answers
81 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
2answers
72 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
4answers
219 views

Electronic laboratory notebook (ELN)

I have been using old style lab book for some time now but with increasing work on computer and storing sequencing results and gel pictures on computer it would be nice to have everything on computer ...
5
votes
1answer
249 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
379 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
188 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
358 views

How do you store membrane proteins?

We're producing some membrane proteins and they aren't amenable to freeze thaws even when we add glycerol. The proteins are solubilized in detergent above the cmc so they should be in micelle form in ...
2
votes
1answer
100 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 anti-...
2
votes
2answers
173 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
200 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
295 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
43 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 ...
2
votes
1answer
42 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?
5
votes
1answer
251 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 ...
2
votes
1answer
32 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 ...
12
votes
2answers
856 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 ...
1
vote
0answers
148 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 ...
7
votes
1answer
1k 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
45 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 ...
-1
votes
1answer
2k 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 ...
2
votes
2answers
663 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 ...
9
votes
1answer
542 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. ...
7
votes
3answers
7k views

What are the advantages and disadvantages of using beta-galactosidase compared to luciferase as a reporter gene?

In the University labs, we have used Beta-galactosidase as a reporter gene to quantify the expression initiated by the stress-response promoter in yeast. This was done by exposing one of the two ...