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

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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 ...
3
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1answer
76 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 ...
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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 ...
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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.
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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
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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 ...
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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 ...
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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 ...
6
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1answer
80 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 ...
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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
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1answer
478 views

For people who work in microbiology labs, what software do you use to manage your strains?

It's just a small microbiology lab that currently records everything on paper, and there's quite few mutants as well. Is Excel commonly used for this sort of thing? Or is there a better software to ...
3
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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 ...
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1answer
5k 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 ...
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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
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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 ...
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 ...
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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: ...
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1answer
113 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 ...
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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?
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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 ...
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?
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0answers
10 views
4
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1answer
92 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
204 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?
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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
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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 ...
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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 ...
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2answers
193 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 ...
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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/ ...
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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 !
11
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3answers
408 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
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2answers
324 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
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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 ...
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 ...
5
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1answer
112 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 ...
6
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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 ...
2
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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
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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 ...
9
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1answer
198 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
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
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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
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4answers
200 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
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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 ...
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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
243 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
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
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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 ...