Questions tagged [lab-techniques]

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

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21
votes
1answer
33k views

What is the difference between HPLC and FPLC and why is FPLC preferable for protein purification?

I've used HPLC (high performance liquid chromatography) before (once, so I'm barely even qualified to know what it stands for) so I was surprised when my labmate told me she would be using an ...
21
votes
3answers
5k views

How long can I store extracted RNA?

If I extract RNA from a (leaf tissue) sample using a one-step phenol:chloroform extraction, how long can those samples be stored at -80°C? And how many times can I defrost and refreeze them before ...
17
votes
2answers
15k views

What effect does vortexing have on a fluid sample that simple mechanical shaking does not?

Some protocols call for fluid samples to be mixed with a "vortexer" on the high setting. What effect does the vortexing have on fluid samples that mechanical shaking does not? Does it shear long ...
16
votes
1answer
9k 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 ...
14
votes
2answers
2k views

Are there issues with filling PCR tubes to capacity?

I'm planning to scale up a PCR reaction, and I'm wondering if filling the PCR tubes to the maximum volume of 200 ul would be a problem. It would mean a lot less pipetting as I would only need 1/4 of ...
14
votes
1answer
92k views

Why do we add salt when precipitating DNA?

All the DNA extraction protocols I have seen involve adding salts to the extraction buffer. What is the purpose of the salts? What happens if they aren't included?
12
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2answers
12k views

Why should I degas my gel solution for polyacrylamide gels?

In protocols for polyacrylamide gel electrophoresis (PAGE) I often see instructions to degas the gel solution by putting it under vacuum for 10-15 minutes before polymerizing the gel. I usually don'...
12
votes
2answers
4k 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 ...
11
votes
2answers
10k views

How do I clean phenol contaminated RNA without losing any of the sample?

I recently extracted RNA from developing plant leaves for the first time, as part of a very long and intensive experiment. The samples were extremely precious because of the amount of effort that went ...
11
votes
3answers
2k 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 ...
11
votes
1answer
5k 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 ...
11
votes
1answer
147 views

Does preservation in ethanol alter leaf litter mass?

I have benthic samples that were collected with an Ekman dredge from some small ponds. The samples contain quite a bit of coarse particulate organic matter (CPOM, basically dead leaves). I would ...
10
votes
2answers
5k views

How do I clean and calibrate pipettes, and how often should I do it?

I work in a lab where all the pipettes are shared. We often have visiting students who come and use the pipettes for a short project. So when I work with them, they might have been handled by other ...
10
votes
1answer
2k 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 ...
10
votes
2answers
966 views

Lab marker for labelling cell culture plastics?

I am a lab manager trying to ask other lab biologists what brand/make of markers their labs use for labeling tissue culture plastics, which need to be repeatedly wiped down with 70% ethanol to ...
9
votes
2answers
1k views

Is working with (nitrile) gloves around a bunsen burner safe?

Recently I have started in a new microbiology lab, and with a new lab come new habits. When I was working with my bacterial (liquid) culture next to the flame (Bunsen burner) wearing nitrile gloves, ...
9
votes
2answers
2k views

Gloves for Cell Culture?

I always wear gloves when I'm doing cell culture. Moreover, I always spray my gloves with ethanol to disinfect, so that I don't contaminate everything. However, I recently heard the argument that ...
9
votes
1answer
2k views

What is the role of glucose in plasmid isolation?

My understanding is that glucose is used in the resuspension solution to prevent cells from bursting by maintaining the appropriate osmotic pressure. Why do we even bother doing this? The cells are ...
9
votes
1answer
1k 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. ...
9
votes
2answers
134 views

Is there a Reverse Transcription optimization for long, 9kb, transcripts?

Has anyone optimized RT for long transcripts (9kb)? The downstream application will be PCR amplification and Illumina library prep. It will be trivial to make internal primers sets for the PCR that ...
8
votes
2answers
182 views

What type of CCD system is required to take photos of luciferase

I'm working with luciferase and I want to be able to take a photo of it. The trouble is, I can see the luciferase glowing in all of its glory in front of me but no matter how hard I try, I can't take ...
8
votes
1answer
6k 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 ...
8
votes
4answers
639 views

Is there a comprehensive life science techniques/methods database?

There are so many techniques/methodologies in the life sciences that we can use to interrogate interesting questions. The thing is, most of us are completely unaware of the available methods we can ...
8
votes
3answers
2k views

Does the Petri dish lid orientation on workbench affect aseptic technique?

When examining an incubated plate, it is rather clear that holding the plate in one hand and the lid in another is best for aseptic technique, or simply resting the lid on the plate as shown below. ...
8
votes
3answers
12k 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 ...
8
votes
1answer
466 views

Optimal pH of protein buffer? Basic principles to adjust buffers according method and analysis

Protein buffers such as PBST, which is used in western blotting, are normally adjusted to pH 7.4. When I try to find why, I find some information about optimal pKa for protein stability. Im not sure I ...
8
votes
1answer
124 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 ...
7
votes
2answers
1k 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 ...
7
votes
2answers
915 views

introduction to Chip Seq

I hope this question is suitable for this site. I am concerned about the Chip experiment part so I think it should be okay. I am a Applied Math student starting to get into bioinformatics and so I've ...
7
votes
2answers
747 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 water ...
7
votes
1answer
5k 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 ...
7
votes
2answers
14k views

How can I resuspend a cell pellet without harming the bacteria?

When using a preculture with Ampicillin in my protein expression, I have to get rid of the preculture medium to avoid carrying over too much beta-lactamases that will destroy the ampicillin in the ...
7
votes
2answers
1k views

Alternatives to CFU plating for measuring number of viable cells?

I am hoping to measure growth rates of a bacterial culture in several growth conditions. I am concerned that these growth conditions may cause cell death, which would lead to a decreased ...
7
votes
1answer
2k views

Alfred Jost's rabbit experiment: how did he actually do it?

I was watching a Crash Course on Biology YouTube video by Hank Green: Great Glands - Your Endocrine System. Hank humorously describes a sex determination experiment by Alfred Jost as follows (...
7
votes
1answer
4k views

How to reduce/eliminate cell clumping in suspension CHO cells, without using an anti clumping agent?

what is the best way to eliminate clumping suspension cells used for transfection. Anti clumping agents interfere transfection and hence can't be used. Though maintaining the cell density low helps, ...
7
votes
1answer
4k views

Role of calcium chloride during competent cell preparation

I am aware of the fact that $CaCl_2$ settles down on the cell wall making it less negative may be by forming bond with Teichoic acid. Also due to the positive charge it attracts DNA (DNA is negatively ...
7
votes
1answer
2k 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
2answers
1k 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 ...
7
votes
1answer
985 views

Can I image Coomassie and GFP in gels at the same time with a fluorescence scanner?

I'm working with a GFP-tagged protein and am routinely using a fluorescence imager (GE Typhoon) and a standard optical scanner to capture fluorescent and absorption images, respectively, of my SDS-...
6
votes
3answers
23k 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?
6
votes
2answers
7k 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 ...
6
votes
1answer
11k views

What are the roles of guanidine-HCl and ethanol in binding of DNA to silica?

I'm trying to understand how exactly the binding to silica gel (in kits) step works and I cannot find any papers which provide an explanation of the physics or chemistry; especially on the way that ...
6
votes
1answer
427 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
votes
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 ...
6
votes
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
votes
2answers
401 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 ...
6
votes
0answers
54 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/...
6
votes
1answer
175 views

EnteroPluri test contamination

An EnteroPluri test has twelve different sectors. It seems odd to me that pulling the inoculating tip from the starting sector (citrate) all the way to the ending sector (glucose/gas) and then ...
5
votes
1answer
6k views

How to avoid air bubbles while pipetting?

I get air bubbles while pipetting small volumes. How can I avoid them ?
5
votes
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 ...