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

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3
votes
3answers
48 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
5
votes
1answer
63 views

Can microdialysis be made in Drosophila melanogaster?

I've asked this before in stack overflow's cognitive science community, and someone recommended me to ask here: I've found a couple of studies using microdialysis on insects, but didn't found any in ...
2
votes
0answers
22 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 ...
10
votes
1answer
106 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 ...
5
votes
2answers
50 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
42 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
180 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 ...
4
votes
1answer
63 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
50 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
51 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
184 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 ...
1
vote
1answer
59 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
89 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 ...
1
vote
3answers
61 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
99 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
39 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
33 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
46 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
24 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 ...
11
votes
2answers
482 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
63 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 ...
1
vote
2answers
115 views

What is the importance of urea in mass spectrometry?

What is the importance of urea in mass spectrometry? We use 8M urea to FASP our proteins prior to mass spectrometry. What is the significance of using 8M urea? and how does it affect the proteins?
6
votes
1answer
126 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
240 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 ...
5
votes
2answers
157 views

Measuring fitness / lifetime reproductive success (LRS) in Drosophila

I am planning a fitness assay of Drosophila melanogaster. I'd like to get a good measure of lifetime reproductive success (lrs) but I don't want to count all the offspring produced over a lifetime by ...
3
votes
1answer
31 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
198 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
250 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
192 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
4k 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 ...
1
vote
2answers
66 views

How to safely sterilize urea-containing growth medium?

I'm using urea-containing growth medium for experiments with bacteria (1-2 l/day). After the experiment, the growth medium has to be sterilized and disposed. I did this so far by autoclaving, but our ...
3
votes
0answers
48 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 ...
3
votes
0answers
27 views

Procedure for doing western blot [closed]

I am writing a step by step guide for doing a western blot for a class. It is intended for any one with basic Biology lab skills. I am hoping people will review my draft and give feedback on how to ...
2
votes
1answer
204 views

Does methanol fixation deform cultured cells?

I use methanol fixation (@ -20⁰C for 10 minutes) when performing immunofluorescence assays on cultured cells. Generally speaking, this results in very good antibody staining. However, the cells tend ...
0
votes
0answers
87 views

Peristaltic pumps in animal perfusion - how important are physiological flow parameters?

Animal perfusion is usually done according to this or very similar procedures. Though not explicitly detailed in this video, the perfusion fluid infused into the Aorta is usually driven via a ...
3
votes
1answer
64 views

Safety of using industrial water for hand washing

Industrial water taps are present in most biology laboratories I have been too. There are accompanied by a sign stating "do not drink". So obviously it is not a good idea to drink it. However I ...
3
votes
1answer
56 views

Well to well variation in thermal cycler fluorescence?

We have an old BioRad ICycler Thermal Cycler with MyIQ single color fluorescent detector. While it's meant for RT-PCR, we've been using it for melt curves and binding assays for different types of RNA ...
2
votes
3answers
533 views

Long term storage of agarose-ethidium bromide gels that have already been cast

I have come up with what I thought was a clever idea: Store the agarose gels I pour, and only cut as many lanes as I need to run later, minimizing wasted agarose (and wasted effort/time) when I need ...
3
votes
1answer
85 views

How can I purify RNA after gel electrophoresis to remove residual acrylamide?

I sometimes use denaturing gel electrophoresis on a preparative scale to purify RNA produced by in vitro transcription. The major issue with this is that the sample after extraction from the gel still ...
6
votes
3answers
3k views

Should I dilute DNA with water or elution buffer?

I've extracted DNA using a kit and final step is eluted with buffer. If I must dilute my DNA samples, do I use the same elution buffer or can I use milliq water?
1
vote
0answers
38 views

How does air-liquid interface (ALI) culture work?

For studying diseases, such as chronic obstructive pulmonary disease (COPD), cells are often grown in air liquid interface. I understand that the common way of establishing this these days is to grow ...
4
votes
2answers
5k views

NaCl role in CTAB - DNA complex in DNA extraction

I have a question about the role of NaCl in the DNA extraction process. So for NaCl concentrations under 0.5M, CTAB and DNA molecules can create complexes. In those concentrations, proteins and other ...
4
votes
1answer
43 views

What's up with all the vague protocols? [closed]

I have lost count of how many protocols I've seen, including those supposed to be professionally written (such as manuals that come with kits from well known brands, or methods sections of papers in ...
1
vote
2answers
930 views

Purifying a linear plasmid after restriction digest?

I expressed a yeast vector in E.coli and purified about 13µg of it. I then linearized it using a restriction enzyme, and attempted to gel purify it. I attempted this twice. The gel showed a clear ...
2
votes
1answer
392 views

How to prepare sample for SDS PAGE?

I usually take my protein sample 0.8ml and sample buffer(2X) 0.2ml for my sample preparation in SDS PAGE. Am I using correct proportion? my protein sample concentration is 4.1mg/ml. What is the ...
6
votes
2answers
322 views

poor RNA quality from zebrafish embryos

Does anyone routinely do RNA isolation from zebrafish embryos? I have embryos from different stages but all below 24hpf. This is the protocol I follow: Take 10-20 embryos Wash once with milliQ ...
1
vote
1answer
107 views

How to separate peptide on SDS PAGE?

I want to separate 1600 Da peptide on SDS PAGE, on 20% GEL I didn't get any band, How can I separate on PAGE? Can I use 25% SDS PAGE?
5
votes
2answers
2k views

Is wiping with RNAse Zap enough to destroy RNAse activity?

From the RNAseZap MSDS, it is an SDS at some unknown concentration, maybe with some NaOH? Some other links suggest there is some NaOH as well. The Ambion site states that RNAseZap destroys RNAse ...
9
votes
3answers
227 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 ...
3
votes
1answer
174 views

Ideal lab glassware cleaner for molecular biologist

I'm using Alconox powder detergent to clean my laboratory glassware, the powder is a pain to store near the sink and to use. Would like to make a concentrated liquid to use in its stead. I have ...