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

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5
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2answers
1k 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 ...
1
vote
1answer
33 views

Amount of reverse transcriptase in µg or mM for qRT-PCR

I am trying to calculate a titration amount for a molecule which I would like to use in my PCR samples. Different molecules have different densities so I would like to calculate the appropriate ...
2
votes
1answer
199 views

Copper for cell incubator to prevent contamination

For some reason the lab seems to have a problem with contamination every so often. It's virtually impossible to prevent bacteria, viruses, fungi, etc. from getting into the incubator every time you ...
2
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0answers
78 views

How long does Lentivirus take to express in vivo mouse neurons?

Does anyone know how long it takes for a standard Lentivirus vector to express its genes (under a strong promoter such as CAG, CB7, etc.), after injection into the brain of a mouse? By hearsay I ...
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0answers
92 views

How is a blood sample analyzed to give the different levels of metabolites?

How is a blood sample analyzed to give the different levels of metabolites? Are there any any procedural differences with each blood test? Thanks in advance!
2
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0answers
146 views

Can I heat Trizol?

I wonder if I can heat Trizol reagent for 30 min 65C. The goal is to disrupt protein-RNA complex while inhibiting nucleases. (I can't use RNasin cause it's inactivated in 65C, and can't use RVC cause ...
4
votes
1answer
103 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 ...
0
votes
0answers
51 views

Western blotting of lung samples

I have been working on lung samples to perform western blotting. I have two related questions about this: I get very nonspecific results: at times nothing at all, at times it glows on membrane but ...
2
votes
1answer
295 views

Strange behavior of a DNA gel

I ran a PCR product of ~300 bp on a 2% TAE-agarose gel for 30 minutes. I used Sybr-safe as a DNA stain. Voltage was 80V. When I imaged the gel, the DNA on the bottom half of the gel, including the ...
3
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1answer
93 views

205 nm UV-Vis readings

Typically we determine the concentration of proteins using a 280 nm reading. However, it is reasonable to use 205 nm. I was curious about the effectiveness of this method.
0
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1answer
514 views

DNA gel extraction: chemical contaminants

I am performing a gel extraction to purify DNA after a double digest with EcoRI and BamHI. After the gel extraction I need to complete a ligation step before bacterial transformation. The problem I ...
4
votes
1answer
1k 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 ...
6
votes
1answer
169 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 ...
4
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0answers
48 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 ...
2
votes
1answer
98 views

What's the difference between a free chromosome fragment and an extrachromosomal array?

This is reference to a review on C. elegans mosaic analysis by Yochem and Herman, in which the authors make a distinction between free chromosome fragments and extrachromosomal arrays. For the ...
2
votes
3answers
832 views

Improving Gel Extraction yields

How can I improve my Gel Extraction yields. We use the standard protocol from Qiagen, gel extraction, dissolve in QG buffer at 42C and purify via anion exchange columns. However, with 500 ng we ...
4
votes
1answer
167 views

What is a simple protocol for staining cells in suspension?

I am an engineering student studying how electric fields affect cells, specifically the phenomena of electroporation in living cells. I know that electroporation is widely used for introducing genes ...
3
votes
2answers
2k 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 ...
6
votes
2answers
527 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
4answers
143 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 ...
2
votes
0answers
108 views

Resuspending Cells from a filter plate

My experiment involves pulling down cells on to a filter plate for a variety of assays. However, I want to ensure that my cells are alive so I am trying to resuspend them so that I can do an accurate ...
4
votes
1answer
190 views

Is DNA green viewer carcinogenic?

I use DNA green viewer in Lab to see DNA and RNA bands. Some peers told me it is carcinogenic and is not safe. Is this correct? If yes, are there better choices to use in working with gel?
2
votes
0answers
17 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
2k 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 ...
5
votes
1answer
325 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
votes
1answer
292 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 ...
7
votes
1answer
351 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 ...
8
votes
2answers
81 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 ...
13
votes
2answers
2k 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 ...
14
votes
3answers
12k 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 ...
8
votes
2answers
145 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 ...
17
votes
3answers
857 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 ...
9
votes
2answers
2k 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
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1answer
15k 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?
8
votes
2answers
4k 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 ...
10
votes
2answers
4k 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 ...