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

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19
votes
1answer
25k 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 ...
19
votes
3answers
2k 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 ...
14
votes
2answers
6k 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 ...
13
votes
2answers
822 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 ...
12
votes
2answers
725 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 ...
12
votes
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 ...
11
votes
2answers
7k 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 ...
11
votes
1answer
45k 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?
11
votes
2answers
7k 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
532 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 ...
10
votes
2answers
4k 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 ...
9
votes
2answers
849 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
330 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 ...
9
votes
1answer
438 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 ...
9
votes
1answer
462 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. ...
8
votes
2answers
163 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
3answers
478 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
2answers
102 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 ...
7
votes
4answers
336 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 ...
7
votes
1answer
1k 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
3answers
6k 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 ...
7
votes
1answer
1k 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
446 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
378 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
600 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 ...
6
votes
3answers
6k 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
451 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 ...
6
votes
2answers
2k 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
2answers
436 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 ...
6
votes
1answer
524 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 ...
6
votes
2answers
2k 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
2answers
222 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
1answer
89 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 ...
6
votes
0answers
73 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 ...
5
votes
1answer
2k views

How to avoid air bubbles while pipetting?

I get air bubbles while pipetting small volumes. How can I avoid them ?
5
votes
2answers
556 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
votes
1answer
165 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 ...
5
votes
2answers
3k 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
votes
1answer
3k 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
votes
1answer
41 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
votes
1answer
192 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
votes
1answer
130 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 ...
5
votes
2answers
729 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
votes
1answer
762 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 ...
5
votes
1answer
74 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
votes
1answer
995 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 ...
4
votes
4answers
212 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
122 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 ...
4
votes
2answers
114 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
votes
2answers
5k 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 ...