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

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2
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1answer
81 views

Plasmid maintenance

I have obtained some plasmids used as integration vectors, this question may apply to all plasmids. I would like to have a somewhat continuos source for these plasmids, let's say that the origin of ...
8
votes
2answers
442 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 ...
2
votes
1answer
47 views

Pippin prep kits - expiration date

Do you use Pippin prep? We would like to buy it, but we need more info. What is the expiration date of kits for Pippin prep? Thanks!
5
votes
1answer
576 views

How to avoid air bubbles while pipetting?

I get air bubbles while pipetting small volumes. How can I avoid them ?
3
votes
1answer
159 views

Benefits of CLARITY?

What are the benefits of CLARITY over this technique that was published more than a year earlier? Of course the second technique needs a fancier microscope that is likely more expensive and requires ...
3
votes
1answer
32 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 ...
1
vote
1answer
52 views

Enzymatic Protein Deglycosylation of TCL

I've done O- and N- linked enzymatic deglycosylation of purified and lyophilized proteins. Following some modifications to the manufacture's protocol, I had great results. I now want a student to ...
10
votes
1answer
21k 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?
5
votes
1answer
57 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 ...
1
vote
1answer
60 views

In vitro transcription, contamination problem

I am using a RNA which is in vitro transcripted before I started my project. It turned out it is not prepared properly and has DNA contamination. Instead of perform the in vitro transcription again, ...
5
votes
2answers
363 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 ...
3
votes
1answer
514 views

Problem with bacterial transformation with electroporation

I have a problem with a bacterial transformation of a yeast gene that I can not solve. I isolated yeast DNA and did a PCR to get my product. I am using pCGCUm vector with a GFP construct. I digest ...
4
votes
4answers
139 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 ...
2
votes
3answers
141 views

Bacterial cell lysis buffer used in proteomics procedures

What kinds of detergent-free bacterial lysis buffers exist? The proteins we're extracting will be later analyzed by LC-MS/MS, and we're looking for a lysis buffer that won't interfere with this ...
2
votes
1answer
111 views

Should the length of the electrodes in the electrophoresis chamber be proportional to chamber's size?

I am trying to build a small horizontal electrophoresis chamber from scratch. I want to use it for comet assay and I will be using only 1 slide, so it's going to be about 3cm wide, 10cm long and 4cm ...
1
vote
0answers
14 views

Gas sampling, in need of large septums [on hold]

I'm designing an experiment that will require many gas samples taken from sealed pipes with a 1" (2.54 cm) diameter. I have been searching for large septums but have had no luck in finding a size ...
6
votes
1answer
293 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 ...
1
vote
0answers
28 views

Improving throughput of CFU plating?

In a separate question I've described my general experimental setup where I need to measure the number of live cells in a growing bacterial culture in a fairly rapid and high-throughput manner. In ...
4
votes
1answer
890 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 ...
1
vote
0answers
94 views

Which is the best way to fix and preserve comet assay slides before staining with Ethidium Bromide?

I want to use the method of Single Cell Gel Electrophoresis (comet assay). I have many samples, but I don't have the appropriate microscope. So I have to preserve my slides until the time comes for ...
2
votes
0answers
46 views

Removal of the Initial Methionine in Venus for FRET

I'm working on building some FRET reporters. In addition to a cleavage site (of varying composition from 15-18AA), a 1 AA linker, I'm using Venus and Cerulean. Initially I was worried that 18AA ...
4
votes
2answers
371 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 ...
17
votes
3answers
1k 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 ...
8
votes
2answers
88 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 ...
3
votes
1answer
61 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
votes
2answers
630 views

How does formaldehyde/PBS or methanol fixation of cells affect lysosomal pH?

The question is fairly simple - does formaldehyde or methanol fixation in preparation for immunocytochemistry/immunofluorescent staining affect the pH of the lysosomes? Some background: I'm trying to ...
2
votes
0answers
135 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
368 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
428 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
1answer
65 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
votes
1answer
119 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
553 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 ...
3
votes
0answers
39 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 ...
6
votes
1answer
193 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
35 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
251 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
97 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
vote
0answers
98 views

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

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!
4
votes
2answers
3k 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
53 views

Western blotting of lung samples [duplicate]

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
106 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
796 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
635 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
2k 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
1k 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
200 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
133 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
58 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
132 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
224 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?