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

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2
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1answer
181 views

Substitute 25mM dNTPs mix with 10mM dNTPs

I need to make a solution of multiple compounds, one of them is dNTPs. The recipe calls for 20 μl 25 mM dNTPs in a 1250 μL master mix. Unfortunately I do not have it available at that concentration, ...
3
votes
1answer
41 views

Changing time and rpm of centifuge

Inspired by this question, I want to ask a question about centrifugation. Suppose a protocol says : 10 min at 2500 rpm . Can we instead centifuge for 5 min at 5000 rpm or 20 min at 1250 rpm ?
4
votes
1answer
10k views

NaCl role in CTAB - DNA complex in DNA extraction

I have a question about the role of NaCl in the DNA extraction process. So for NaCl concentrations under 0.5M, CTAB and DNA molecules can create complexes. In those concentrations, proteins and other ...
3
votes
0answers
191 views

Gibson assembly using NeBuilder

I am supposed to construct a plasmid that contains features from two other plasmids. My strategy is to generate three fragments form the two other plasmids. I was encouraged to try Gibson assembly, ...
2
votes
1answer
115 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 ...
9
votes
2answers
859 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
68 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
2k views

How to avoid air bubbles while pipetting?

I get air bubbles while pipetting small volumes. How can I avoid them ?
3
votes
1answer
339 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 ...
4
votes
1answer
44 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
133 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, ...
4
votes
1answer
561 views

For people who work in microbiology labs, what software do you use to manage your strains?

It's just a small microbiology lab that currently records everything on paper, and there's quite few mutants as well. Is Excel commonly used for this sort of thing? Or is there a better software to ...
6
votes
2answers
437 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 ...
4
votes
4answers
213 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
410 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 ...
3
votes
1answer
969 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 ...
1
vote
0answers
54 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 ...
6
votes
1answer
527 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 ...
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 ...
1
vote
0answers
164 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 ...
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 ...
3
votes
1answer
64 views

Non-fatal / low-harm measurement of Drosophila traits

I am trying to generate some candidate traits to measure in a fitness assay of wildtype outbred (lab population) flies. The key trait I will measure is lifespan. I am looking for some additional ...
1
vote
1answer
108 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 ...
2
votes
1answer
212 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 ...
2
votes
0answers
57 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
1answer
91 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 ...
8
votes
3answers
482 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. ...
3
votes
2answers
1k 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 ...
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, ...
2
votes
0answers
227 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 ...
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
2answers
731 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
190 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 ...
3
votes
1answer
1k 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 ...
2
votes
1answer
60 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?
3
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0answers
40 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 ...
4
votes
1answer
73 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?
4
votes
2answers
764 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 ...
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 ...
1
vote
1answer
41 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
388 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
155 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 ...
4
votes
1answer
400 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
2answers
315 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 ...
1
vote
0answers
57 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 ...
2
votes
1answer
703 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
votes
1answer
142 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
votes
1answer
1k 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
3k 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 ...
7
votes
2answers
382 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 ...