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

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4
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2answers
152 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
50 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 ...
1
vote
2answers
69 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
56 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
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3answers
76 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
38 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
30 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
32 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
20 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 ...
10
votes
2answers
420 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
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0answers
50 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
70 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 ...
1
vote
2answers
84 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
78 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
134 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
148 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
28 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
122 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
215 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
149 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
45 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
38 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
23 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 ...
1
vote
1answer
138 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
57 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
59 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
51 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
370 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
74 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
2k 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?
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0answers
28 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
4k 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
39 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 ...
0
votes
2answers
653 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
218 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 ...
5
votes
2answers
301 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
81 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
191 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
163 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 ...
6
votes
2answers
548 views

Alternatives to trypsin for cell detachment?

I have ran out of trypsin and need to passage my cells (immortalized chondrocytes, C28/I2) today or tomorrow. I have been out of town and forgot to order more trypsin. I was wondering if there are ...
0
votes
1answer
31 views

Does adding antibiotic after 5-10 mins of innoculation affect the protein yield or growth?

I've asked a lab colleague the same question. She said, it would loosen the bacterial cells in the LB medium and plasmids would come out. Is that true? and why?
5
votes
2answers
157 views

How do you visualize RNA on a gel?

I have run an in vitro transcription reaction and produced some RNA of a single species and definite size. I would like to visualize it to check if the reaction worked. Can I do this on an agarose ...
8
votes
1answer
263 views

How do I put on a labcoat?

Let's assume I am wearing a labcoat for two reasons: Prevent the various bacteria, proteins, skin cells and substances on my clothes and my skin from contaminating my experiments. Prevent various ...
4
votes
1answer
41 views

Effect of Ethanol on Brain Volume Measurements

I want to compare the brain sizes of two populations of fish. However, all the samples I have were fixed in 95% ethanol. As far as I know, 4% formalin is the normal fixative for soft tissues. Can I ...
4
votes
2answers
84 views

Short, concise, practical manual for doing experimental biology

I am am physical scientist working in biology, and have recently started doing experiments. I would like a manual akin to "Numerical Recipes", but for the lab: straight forward, easy instructions on ...
2
votes
1answer
4k views

How does a TOPflash/FOPflash assay work to detect beta-catenin protein expression?

I am reading an article (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3596711/) wherein a TOPflash/FOPflash assay is used to detect beta-catenin protein levels in a COS-7 cell line. I can't find a good ...
2
votes
0answers
103 views

Origin of the 260/280 ratio?

This is not a duplicate of all the other 260/280 ratio questions, I already know that DNA is supposed to be 1.8 and RNA is supposed to be 2.0. However, this might be more appropriate for chemistry, ...
4
votes
0answers
55 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 ...