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

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4
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0answers
179 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, ...
2
votes
0answers
115 views

What tests can be performed to test the purity and quality of the raw peptide HCG (Human Chorionic Gonadotropin)

What tests could be run to test the purity and type of HCG? We are looking to purchase HCG from China but the purity and quality varies between labs, we are able to receive samples of the raw peptide ...
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 ...
5
votes
1answer
324 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
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1answer
63 views

How can I save bacillus strains on filter paper without an -80 degree freezer?

I want to save my bacillus strains but I don't have access to a -80 degree freezer. What are possible alternatives?
3
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1answer
95 views

How do different tissue culture matrices affect background in fluorescent microscopy?

In response to my previous question, I've been reading up a little bit on poly-D-lysine, Collagen I, Collagen IV, laminin, and other tissue culture coatings that promote cell adhesion. I've always ...
2
votes
1answer
426 views

Optogenetics - How do microbial opsins work?

I'm just introduced to the optogenetics method and am having some trouble grasping the genetics (of the optogenteics) part of things. So we have Retinal and Opsin that form Rhodopsin molecule that ...
0
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0answers
31 views

Ovalbumin Detection

Is there a practical way to detect whether or not a baked good has egg in it? I'm thinking of something that can be brushed on to evolve a color change. Any help is appreciated.
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0answers
61 views

What material is recommended for autoradiography cassettes when working with 32P?

So, during radiation safety training, much hullabaloo is made regarding how the high energy beta emitted by 32P produces Bremsstrahlung radiation when striking a high Z shielding material. As such, ...
3
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0answers
37 views

Can I purify polyhydroxyalkanoates by heating the cells extensively?

Traditional methods of purifying polyhydroxyalkanoates (PHAs) and other bioplastics made by bacteria involve washing the cells with harsh chemicals or strong bases.I'm interested in maintaining the ...
1
vote
0answers
40 views

How can I view modENCODE data faster?

I am trying to view several data tracks in the modENCODE GBrowse genomic browser. However, the site is so slow, it is practically unworkable. Is there a faster way to explore the data?
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 ...
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
votes
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 ...
1
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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!
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 ...
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 ...
3
votes
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.
6
votes
2answers
524 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 ...
0
votes
1answer
513 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 ...
2
votes
3answers
826 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 ...
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 ...
4
votes
0answers
47 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
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
189 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 ...
7
votes
1answer
350 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 ...
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 ...
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 ...
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 ...
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 ...
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 ...