Questions tagged [lab-techniques]

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

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6
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2answers
4k views

How long can I store autoclaved disposables and reagents?

I want to stock up on autoclaved disposables, such as Eppendorf tubes, pasteur pipettes etc., and some buffers. If I don't open the container after autoclaving, how long can I store autoclaved ...
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1answer
2k views

What are good practices with reusing desalting columns

At least according to a few sources Prozyme and Protocols-Online, it is possible to reuse desalting columns and since I'm cheap I would like to also. Key things seem to be washing with several column ...
6
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2answers
420 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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0answers
76 views

What is the fastest way to crystallise lysozyme (for student course)?

High school sudents are going to visit my university and I plan to demonstrate crystallisation of lysozyme. I ordered pure lysozyme from VWR. I can easily crystallise this within 15 min in batch (4% w/...
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3answers
164 views

Saturated Mutagenesis Screening

I am hoping to mutate the active site of the enzyme I am researching that has 5 residues in proximity with the substrate. I am wondering how many colonies I'll have to assess to theoretically sample ...
5
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1answer
9k views

How to avoid air bubbles while pipetting?

I get air bubbles while pipetting small volumes. How can I avoid them ?
5
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3answers
253 views

Double Digestion with Restriction Enzymes Using Different Buffers

I am currently working on preparing a 9 kb sequence of DNA for restriction digestion into the pBAD30 expression vector. There are very few restriction enzymes that do not have a restriction site ...
5
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2answers
3k 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 ...
5
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1answer
608 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 ...
5
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1answer
16k 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
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1answer
921 views

Are vacuum centrifuges the same as normal centrifuges?

I came across a method that used a vacuum centrifuge: Carvalho et al. 2012. The diversity, antimicrobial and anticancer activity of endophytic fungi associated with the medicinal plant ...
5
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1answer
96 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 ...
5
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2answers
559 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 ...
5
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2answers
4k 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 ...
5
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1answer
9k views

What is immunopanning (vs. immunoprecipitation and FACS)?

I had never heard the term before today. From what I can tell, it's using antibodies to purify a cell population of interest. I would appreciate more details, especially in how it differs from "...
5
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2answers
18k 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 ...
5
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1answer
6k views

Basic step by step methods for PCR & Gel electrophoresis class

I'm teaching a class how to do PCR & gel electrophoresis soon and would like it if you could check through my basic step by step instructions - it's a while since I've done one. Is there anything ...
5
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1answer
67 views

Autoclaving media Question

1.When I autoclave media should I close the cap tightly or slightly? I put all tools in plastic bag when I autoclave, should I close slightly the plastic and when it finish I close tightly the ...
5
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1answer
229 views

What kind of “size” does SDS-PAGE separate by?

What is the exact property of the protein that affects migration of bands in SDS-PAGE; is it length, volume, or mass? For example, will GGGGGGGGGG (glycine 10-mer; ...
5
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1answer
822 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 natural ...
5
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1answer
61 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
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1answer
21k 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 ...
5
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2answers
1k views

High Current (Speed) Transfer Buffer Recipe

Does anyone know an effective buffer mix to use for high current Western transfers? We are successfully using the vendor's premixed buffer to transfer a wide range of protein sizes to PVDF membranes ...
5
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2answers
2k views

Does bleach destroy RNAse activity, and if so, how does it do it?

I am working with RNA samples, and I'm trying to be very careful about RNAse contamination. I have some questions about bleach, though. Some people say that a solution of bleach is enough to destroy ...
5
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1answer
45 views

How standardized is lab-grade skim milk?

I was surprised to learn in the answers to this question that in many cases the preferred cryoprotectant for many organisms is skim milk, rather than something more simple and well-defined like ...
5
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1answer
32 views

Is there a biological or technical reason for a 40 Ct cutoff in nucleic acid detection assay?

I have developed and validated a modified a nucleic acid test (NAT) for SARS-COV-2 detection using real-time RT-PCR (aka rRT-PCR, aka RT-qPCR). My assay is not for diagnostic use, but for donor ...
5
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1answer
1k 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 ...
5
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1answer
2k views

Using ion-exchange chromatography to purify DNA from a cell extract - Is DNA more negatively charged then RNA?

When applying this method we have a glass or plastic column of resin which is positively charged. Then we pour cell extract into the column in order to capture the negatively charged particles which ...
5
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1answer
100 views

What method would you use to genotype SNPs in low quality samples?

What method would you use to genotype SNPs in low quality samples? I ideally want to genotype hundreds of SNPs in hundreds of scat samples (very low amount of target DNA, potentially degraded and ...
5
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1answer
150 views

Optimization of Signal Peptides

We are looking at modifying the signal peptide of a receptor in a common immortalized human cell line. The cell line already expresses an unusually high amount of the protein, but much of it is not ...
4
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2answers
4k 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 ...
4
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1answer
114 views

Alternative dyes for Gram staining

In the Gram stain, is there any replacement for primary stain, secondary stain, decolourizer and mordant? What results will the replacements produce? I found that crystal violet can be replaced with ...
4
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2answers
249 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 ...
4
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4answers
297 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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3answers
92 views

Book recommendation: protocols and recipes handbook for molecular biology / biochemistry

I'm a 5th-year PhD student in chemical biology. I've mostly been doing computational work, so my bench skills are rusty. To help me, I'd like a handbook of common techniques -- transformations, ELISA, ...
4
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2answers
156 views

What is an Ihh-/- mouse?

This one is too basic question: I just came across $Ihh^{-/-}\ $ mouse. Is that means this mouse devoid of that gene Ihh. What is this sign called and are there other such representations?
4
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1answer
3k 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?
4
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2answers
375 views

Why do protein solutions have to be alkalised in biuret test?

I’ve read that CuSO4 solution reacts with peptide bonds that connect amino acids to create a violet colour, but the instructions always tell me to add NaOH solution to the protein solution before I ...
4
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1answer
209 views

How do you keep track of past/new lab protocols?

Does there exist a list grouped my subject (e.g. 'microscopy') for keeping track of what protocols have been designed and used? Closest I could find is http://www.protocol-online.org/ but it's fairly ...
4
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1answer
11k views

How to convert skin temperature to core temperature?

As opposed to regular invasive thermometers, there are non-invasive IR thermometers to measure the temperature. For example Thermofocus. I read in many sources that these IR thermometers only measure ...
4
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3answers
5k 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 ...
4
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1answer
388 views

Why is sorbitol used in buffers?

Many protocols in my lab use sorbitol in buffers. For instance, in co-immunoprecipitation, we include it at a final concentration of 200 mM in our lysis buffer. I'm not entirely sure why though. I ...
4
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1answer
114 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 ...
4
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1answer
54 views

Prevalent large (>=90kDa) maintenance protein/loading control

I was wondering if anyone had recommendations for good, large (hopefully 100kDa+), control proteins that would be present in most mammalian cells. I'm working mostly with tissue samples from humans ...
4
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1answer
124 views

What makes drosophila eyes red? and is it stable?

I have Drosophila melanogaster which I am doing an eye pigmentation assay on in the future. To do this I will dissolve the heads, 10 of them removed from frozen whole flies, in acidified ethanol for ...
4
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2answers
1k 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 ...
4
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1answer
149 views

DNA quantification in a high school bio lab

I'm working on a project in a high school bio lab (so limited resources), and I need a way to quantify the concentration of DNA in a PCR product. I can't use spectrophotometry (cheap ...
4
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2answers
2k 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 ...
4
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2answers
566 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 ...
4
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3answers
6k views

DpnI over-digestion

We have a long protocol that we are optimizing that includes DpnI digestion of a PCR product (to remove any of the template DNA if it's methylated, and while we're not certain in the blind tests, ...

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