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Questions tagged [lab-techniques]

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

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5 votes
4 answers
2k views

LCMS/MS versus Western Blot

I have a general question regarding which method would you recommend me to use if I would like to investigate the difference in the level of several proteins in tissue samples and compare different ...
3 votes
0 answers
29 views

How can I non-destructively blind/cover one drosophila eye for behavioural assays?

I am looking for a protocol for non-destructively covering one eye of Drosophila for a behavioural and subsequent imaging experiment I am planning. I am aware of using wax to paint over the entire eye ...
6 votes
1 answer
58 views

dilutions in bacterial culture

In overnight bacterial culture, why do we put bacteria in a small flask to culture before moving it to a larger flask? Why not just put the sample in a large flask to begin with? This is in reference ...
0 votes
0 answers
10 views

What are ways to measure carbon fixation of microalgae?

So I am conducting a very simple experiment about microalgaes, particularly their biofixation of carbon dioxide. In a small compartment, where microalgaes are suspended in water with an air pump that ...
0 votes
1 answer
47 views

Extremophile hunting and identifying unknown microbes

I'm going extremophile hunting! We're doing a trek by boat between some islands with semi-active volcanic terrain to try to document some interesting species, and running into a problem I've never ...
3 votes
2 answers
297 views

How should I measure the oxygen dispersed during photosynthesis in pondweeds?

I am to conduct a lab investigating how different wavelengths of light affects photosynthesis in Egeria pondweeds. The idea is to put color filters on light bulbs and shine them on the pondweed in a ...
0 votes
1 answer
68 views

How come SSBPs in RPA don't bind primers?

I've started reading about the recombinase polymerase amplification (RPA). I'm learning that in RPA, recombinase enzyme binds to primers, then makes them anneal to the complementary target DNA strand, ...
1 vote
1 answer
68 views

Can single-Use Gloves carry bacteria on their outer surface?

In bacteriology, can I touch items (e.g. oxidase discs) with my gloves not fearing to contaminate these items? Are these gloves (e.g. Disposable nitrile gloves) made so that bacteria can't adhere to ...
4 votes
2 answers
400 views

Primer design for site-directed mutagenesis

In our practical course about modern cloning methods, we performed point mutations on a promotor via site-directed mutagenesis. As far as I understand that method you need forward and reverse primers ...
0 votes
1 answer
97 views

Where can I find the standard curves for Bovine Serum Albumin (BSA) and ovalbumin as determined through the Bradford Assay?

I was wondering if anyone would please be able to point me in the direction of a database or something similar which contains standard curves/absorbance levels for BSA and ovalbumin, as measured using ...
2 votes
0 answers
123 views

Immunoaffinity chromatography: avoiding damage to the antibodies from proteases

What are the possible methods to prevent the digestion of antibodies (mainly Polyclonal) by proteases during affinity chromatography? I read some papers about doing modifications to the anitbodies: ...
0 votes
0 answers
55 views

Methods of denaturing BL21 E.coli other than using lysis buffer and sonication

The goal is to try different denaturing methods for BL21 E.coli. However, finding detailed paper on this is challenging as most articles do not seem to include the details of their denaturing ...
1 vote
2 answers
867 views

Is there any reason to use an upright microscope over an inverted microscope?

I've never actually owned an inverted microscope, but it seems it has only advantages compared to an upright microscope: taller, heavier samples; no crashing the objective into the glass slide; easier ...
2 votes
0 answers
43 views

chip sequencing

I understand the concepts of steps in Chip-seq up to DNA purification, but I don't get how one can then amplify the purified DNA samples that are once bound to the proteins.... Since sequence-specific ...
1 vote
1 answer
97 views

What are some experimental techniques to identify binding partners?

What experimental techniques can be used to identify binding partners for a given protein? I know that if you have some candidates that may bind to a given protein, then you can use techniques such as ...
1 vote
1 answer
4k views

RNeasy Mini Kit low 260/230 ratio -- can I purify this RNA for further use?

I used Qiagen's RNeasy Mini Kit to isolate RNA from 5*10^5 C28/I2 (immortalized human chondrocytes). However, my yield is low (~25 ng/ul), but my 260/280 ratio is great (~2.3), and my 260/230 ratio is ...
6 votes
1 answer
784 views

Origin of the 260/280 ratio?

This is not a duplicate of all the other 260/280 ratio questions, I already know that DNA is supposed to be 1.8 and RNA is supposed to be 2.0. However, this might be more appropriate for chemistry, ...
2 votes
1 answer
85 views

Why is my tissue sample coagulating during centrifugation? How can I prevent this?

I am working on extracting water-soluble compounds from a biological tissue sample. A problem I am encountering is that the sample is too cloudy with debris/large tissue pieces to filter into HPLC ...
0 votes
0 answers
173 views

What nutrients are necessary for Bacillus subtilis germination?

What are the necessary conditions to germinate Bacillus subtilis spores? Under laboratory conditions, spores of B. subtilis are often heat-activated for 30 min at 70°C to increase their germination ...
2 votes
1 answer
289 views

How does SDS-PAGE separate based on mass?

In SDS-PAGE, electric field and mass-to-charge ratio are approximated to be constant for all proteins. Also, if $F=qE =ma$, then $\frac{m}{q}a=E$. Thus, all proteins must migrate with a constant ...
2 votes
0 answers
101 views

How to prepare ethanol-preserved insects for imaging using SEM

I have aquatic insect larvae (soft-bodied) currently preserved in 70% ethanol. I would like to image these using a scanning electron microscope (SEM). I am wondering whether I need to/can start at ...
3 votes
0 answers
30 views

What colors I am looking for in the TSA stain in this paper?

I'm just having a little bit of trouble and want to make sure that I am seeing the same things that the original researchers were seeing. This is the name of the paper Hair Cycle Control by Estrogens: ...
6 votes
1 answer
238 views

DNA quantification in a high school bio lab

I'm working on a project in a high school bio lab (so limited resources), and I need a way to quantify the concentration of DNA in a PCR product. I can't use spectrophotometry (cheap ...
2 votes
1 answer
90 views

Why is cDNA usually lacking in terminal sequences of the template mRNA?

It appears from presentations I have attended that cDNA often lacks terminal sequences (usually 5′) which have not been copied from the mRNA. This puzzles me a lot, but I have not been able to find ...
1 vote
1 answer
194 views

Sabouraud dextrose agar breaking?

I recently started growing some Geotrichum Candidum (GC) on Sabouraud dextrose agar (SDA) beds. The SDA was poured into sterilized petri dishes. After setting the plates in a cooler (20 C) for a day, ...
4 votes
1 answer
205 views

Spectroscopic methods for quantifying peptides/proteins with or without Tryptophan or Tyrosine content

I have several peptides (20-50 amino acids long) which I want to quantify the solubility/concentration in a solvent at certain temperature and pH. These peptides may or may not contain Tryptophan or ...
0 votes
1 answer
134 views

What is the best way to stack parafilm wrapped petri dishes?

I have several petri dishes that are individually wrapped with bemis parafilm. As space is somewhat limited, I've begun stacking them on top of each other, however in doing this the wrapping for each ...
3 votes
0 answers
40 views

Lab device to periodically, automatically transfer zooplankton between containers

I need a device to periodically draw a sample of transfer small aquatic organisms from one container to another without crushing/killing them. Each sample would be on the order of 10ml. My sketch of ...
1 vote
1 answer
110 views

Has anyone who has ever isolated synaptosomes using subcellular fractionation before know what the 'crude/heavy membrane fraction P2' is?

I am reading a journal paper where they analyse the proteome of synaptosomes. In this paper, they isolate synaptosomes from the hippocampi of mice. I know that synaptosomes contain the complete ...
5 votes
1 answer
943 views

Safety of using industrial water for hand washing

Industrial water taps are present in most biology laboratories I have been too. There are accompanied by a sign stating "do not drink". So obviously it is not a good idea to drink it. However I ...
0 votes
2 answers
2k views

How long is agar agar good for?

I'm a high school science teacher, and am starting at a new school. I'd like to do some labs with bacteria and so I'll need to make plates with agar agar. I found 3 huge jugs of powdered agar in the ...
1 vote
1 answer
294 views

Transformation and PCR in molecular cloning

After obtaining the recombinant DNA, it is common to transform into E. coli to screen for recombinant DNA and amplify it. But I would like to ask can we amplify it using PCR? I think there will be 3 ...
3 votes
0 answers
283 views

What are the safety precautions I should take if I want to experiment with C. elegans (at home)?

Caenorhabditis elegans (C. elegans) is a nematode (roundworm), and many nematodes do infect humans and cause diseases. How much is C. elegans safe (or unsafe) in this respect? C.elegans is a popular ...
1 vote
1 answer
997 views

What is the difference between 4th generation sequencing and NGS?

The generation of sequencing technologies has come on leaps and bounds and there are stark differences between the types of technology used. There is a great Q&A here What is the difference ...
4 votes
1 answer
128 views

Why would this viral strain-specific antiserum fail to immunoprecipitate the same (98% identical protein) from another strain?

I'm reading this paper https://www.ncbi.nlm.nih.gov/pmc/articles/PMC392475/ and I can't work out why a certain immune serum didn't work on the same viral protein but from different strains. The serum ...
-1 votes
1 answer
70 views

What antibody targets are being tested for in the publicly offered UK antibody test?

In late August 2021 the NHS (UK) offered people who test positive for COVID what is referred to in this BBC report as a “new antibody test”. However, I have been unable to find out what exactly is new ...
1 vote
0 answers
26 views

How do I cover Cellstrainers?

I‘m doing some experiments for my bachelors thesis and I‘m using some Cell strainers by Roth for it (https://www.carlroth.com/com/en/accessories/cell-strainer-easystrainer™/p/cly9.1). My problem is ...
12 votes
1 answer
382 views

What's happening in the "C" and "T" stripes of a covid test kit?

I have a COVID home test kit which produces C and T (control and test) stripes when the solution is applied to the strip. Something similar happens in pregnancy test kits. I understand the purpose of ...
9 votes
2 answers
493 views

Tips for longer fragment size and higher purity of insect DNA

Aim In a pilot experiment I tested three different genomic DNA extraction methods on the non-model organism Pieris mannii (southern small white; Insecta, Lepidoptera, Pieridae). The goal was to ...
5 votes
0 answers
56 views

Methods for getting clean C. difficile spore preps without spores clumping together

I'm a microbiologist recently who recently started working on an ongoing C. difficile project for the first time. One of the things I need to do is verify the lower detection limit of my qPCR ...
3 votes
1 answer
101 views

Isolating colonies in a pure culture

I want to conduct antimicrobial susceptibility tests for which I've to culture bacteria first. Since I've bought KWIK-STIKs, (KWIK-STIK contains a single lyophilized microorganism which have to be ...
2 votes
1 answer
60 views

Washing buffers for protein identification?

I am currently learning protein identification techniques and come across ELISA and Western Blot. In these methods, a washing buffer is required to wash out the antibodies that are not bound to the ...
2 votes
1 answer
90 views

Procedure of diagonal electrophoresis

I am learning protein sequencing and it seems that diagonal electrophoresis is a common method in the identification of disulphide bonds (S-S) exist between polypeptides in a protein. Questions: ...
3 votes
1 answer
817 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 ...
1 vote
1 answer
85 views

How to identify plasma cells that only produce monoclonal antibodies?

I am studying the procedures of forming hybridoma cells for generating a large number of monoclonal antibodies. Before the procedure of fusion (with multiple myeloma cells) happens, I would like to ...
6 votes
2 answers
97 views

Which method of gene amplification for toehold switches?

My team and I are from a high school and are planning to carry out some research investigating some toehold switch riboregulators which we have designed in silico. However, we have little experience ...
0 votes
1 answer
83 views

Is there a scale suitable for continuous tracking and recording of plant weight?

I want to measure water transpiration and evaporation in a bonsai tree, and measuring the weight of the tree in its pot is my proxy for water usage. This has worked well with manual measurements, but ...
0 votes
1 answer
125 views

Enzyme inhibitor leads to higher turnover rate?

I'm currently working on a project where I have to deal with enzyme inhibition. The purified enzyme shows a good substrate turnover. When I try to inhibit it with different inhibitors described in ...
1 vote
1 answer
97 views

Can GFP reporting be used to track localization of peptides in the ER, Golgi, and plasma membrane?

Suppose I want to study the trafficking of a peptide throughout the ER, Golgi, and plasma membrane. An idea I had was labeling a secreted or plasma membrane integral protein with GFP and using time-...
0 votes
0 answers
420 views

How can I clone a gene into a plasmid vector with an N-terminal his tag and TEV cleavage site between the tag and the start of the sequence?

I'm a scientist who has significant experience in chemistry but am relatively new to molecular biology and biochemical techniques. I'm trying to make an isolated domain of a protein (166 residues, 19....

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