Questions tagged [lab-techniques]

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

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Alternatives to Matrigel in chick aortic arch assay

When conducting a chick embryo aortic arch assay in order to see angiogenesis inhibition properties of various substances(originally in powder form but soluble in water or ethanol) is it possible to ...
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186 views

Using nanodrop for analysing biological samples other than nucleotides

I am a 3rd timer postdoctoral fellow with some experience in molecular biology and biochemistry, but major skills in Zoology and Natural History. I am studying some natural extracts, and isolating ...
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3answers
542 views

Getting PCR amplification at annealing higher than Tm!

I am amplifying a gene where in a gradient pcr i am getting amplification at an annealing temperature about 5 degrees (67) higher than Tm (62.5)? What is wrong here? Also, I am getting a very strong ...
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141 views

Killing microorganisms on agar plates?

If I grow microorganisms on agar plates and I expose them to UV light. How exactly will I know that the microorganisms have been killed? I was watching a video that showed spots in the petri dish ...
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17 views

what are best methods for mrsa screening in hospital?

methods for screening MRSA are based on PCR (mecA detection) and MRSA latex test and culture. I need to know sensitive, specific and cost-effective methods for diagnosis MRSA infection on one work day ...
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2answers
185 views

Why do protein solutions have to be alkalised in biuret test?

I’ve read that CuSO4 solution reacts with peptide bonds that connect amino acids to create a violet colour, but the instructions always tell me to add NaOH solution to the protein solution before I ...
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54 views

I did an agar gel electrophoresis but I could not see DNA, why? [closed]

After performing a PCR, I ran the products (700 bp) in an agar gel electrophoresis along with a Genedirex ladder (100 bp), but the transilluminator did not reveal them.
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1answer
53 views

Immunoprecipitation compared to western blotting

Immunoprecipitation and western blotting are both used to locate a specific protein within a sample and to isolate it. In immunoprecipitation, a specific antibody and agarose beads ( or other ...
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2answers
34 views

Preparing sample for SDS PAGE

I have more than 10 cell lysate samples (70 µL each) whose concentration varies from 1.9 mg/mL to 4.8 mg/mL. I have 5X and 2X SDS sample buffers. I would like to prepare SDS PAGE samples in such a way ...
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16 views

How do I homogenise a small pellet of 10 to 50 cells within less than 50 µL?

I have a problem with declumping/ homogenising bacterial cells in small volumes (10 to 50 µL). I know that I can get all of my cells out of the tube, but they are still stuck together and won't form ...
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1answer
37 views

How many cells should be seeded for passaging?

Do you count cells when you passage them? I found that many people will according to a ratio (maybe 1:2 or 1:3,etc.) to subpackage. It is so indistinct! What is the optimal seedling density or ...
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9 views

Re-stain slice using a different secondary antibody?

I stained a free-floating ~300uM brian slice using 4 different 1º antibodies (rabbit, chick, mouse, rat). For the mouse primary, I mistakenly used an Alexa-350 secondary, when I should have used 594. ...
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How to isolate a high proportion of available helminth ova from animal faeces/feces without damaging them?

I need to isolate large numbers (tens to hundreds of thousands) of cat (and later dog) hookworm ova from cat/dog faeces. I have a centrifuge, and various sieves with pore sizes of 20, 63, 75, 106, ...
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1answer
18 views

How to supply oxygen to a culture vessel?

I'm reading this paper: https://www.jstage.jst.go.jp/article/jpsa/51/3/51_0130043/_pdf From day 17 of the culture period until hatching, pure oxygen was supplied at a flow rate of approximately 500 ...
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1answer
75 views

Must I HPLC-purify my PCR Primers for amplicon sequencing with PacBio SMRT?

I would like to order some primers for amplicon sequencing. I am using universal tag primers (unusually long 30-mers recommended by PacBio for SMRT, both F&R) + barcodes (16-mers). The resulting ...
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1answer
103 views

Minimal viable EEG equipment for dissertation research on BCI / BMI

I am planning out a dissertation study of Brain-computer Interface (a.k.a. Brain-machine Interface, BCI, BMI, etc) applications. One of the 3 papers in that dissertation will involve collecting ...
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19 views

Why both forward and reverse primer are added in asymmetric PCR?

If the purpose of Asymmetric PCR is to amplify single targeted strand of dsDNA then instead of adding just one primer for targeted strand why do we add both forward and reverse primer? Upon addition ...
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51 views

What is the fastest way to crystallise lysozyme (for student course)?

High school sudents are going to visit my university and I plan to demonstrate crystallisation of lysozyme. I ordered pure lysozyme from VWR. I can easily crystallise this within 15 min in batch (4% w/...
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Does the preservation fluid (ethanol or formaldehyde) leach heavy metals from mollusc tissue?

In a thread on researchgate, the concern was raised that long term exposure to unbuffered preservation fluids such as ethanol or formaldehyde may leach heavy metals from biological samples due to a ...
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3answers
103 views

Double Digestion with Restriction Enzymes Using Different Buffers

I am currently working on preparing a 9 kb sequence of DNA for restriction digestion into the pBAD30 expression vector. There are very few restriction enzymes that do not have a restriction site ...
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1answer
1k 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 ...
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1answer
29 views

How to confirm secondary structure formation of Precursor miRNA on gel?

I want to fold precursor-miRNA into its secondary structure and then confirm it on gel. At first, I heat it in annealing buffer at 95°C for 5 minutes and cool down slowly. Then I run it on gel but the ...
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32 views

Is it normal to see yeast cells stacked in the Z axis when counting yeast?

I just got started with yeast cell counting and was surprised to observe yeast cells that are at different z axis depths at 400x. That is, by adjusting the focus (not the x/y stage) I can make some ...
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What is the connector called to attach a luer-lok fitting to a catheter-tip syringe?

I make 3D printer heads that accept syringes for depositing liquids and emulsions. We've made a 150cc syringe holder, but we can only find these syringes with a catheter type tip, and we'd like to ...
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Thermophilic Total plate count with PCA with Triphenyl tetrazoliumchloride as supplement

Hello I'm am a fourth year microbiology analyst and I'm busy with finishing my final internship. During my final internship I have to complete a own assignment. the assignment I got was to find out if ...
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1answer
8k views

When running gels what is the difference between constant volts or constant amps?

In general, you want to be consistent with running your gels either at constant volts or constant amps. However, it is very clear that during the progression of both PAGE and agarose gels, the free ...
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1answer
42 views

Improving DNA quality and yield from stool samples

I prepared genomic DNA from pig stools but the concentration was very low — about 3~4 ng/µl, when I measured it using nanodrop. The A260/A280 and ...
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1answer
52 views

Why pellet and resuspend E. coli for plasmid prep

For maxipreps, cant you just add all the stuff that would be in P1 (RNase A, EDTA) then just add P2? Because if we pellet cells then resuspend theres got to be a reason, right? Is it slats and other ...
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1answer
47 views

Why electroporator need a cuvette?

I have never obtain an Electroporator for my home-lab. Because it's very expensive, I'm trying to create a diy cheap version for myself. In all commercial design, I see that we need to put the sample ...
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1answer
123 views

How to perform cell biology research without lab affiliation [closed]

Related to this question, I am wondering just generally how one can do cell biology research without being part of an already well-established University laboratory. I am not doing this myself but am ...
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1answer
146 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 ...
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1answer
2k views

What is the role of glucose in plasmid isolation?

My understanding is that glucose is used in the resuspension solution to prevent cells from bursting by maintaining the appropriate osmotic pressure. Why do we even bother doing this? The cells are ...
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1answer
70 views

How does the OD correlate with the number of algae cells?

My research team and I are using a spectrometer to measure the OD of an algae sample. We are trying to answer the question "Is there a correlation between OD and the number of algae cells?" What ...
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111 views

Why do Petri dish lids not fit?

I understand that they are generally "vented", but why such a poor fit? For the batch I bought, the lid and container don't even recognizably go together (like 1/2" difference in diameter). The wind ...
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50 views

What kind of sieve does one use to extract meiofauna?

Suppose I have a core of sediment, and I want to extract the meiofauna from it, in order to study these organisms. According to the sources I could find, this involves using a very fine (e.g. 45 μm) ...
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41 views

DNA preservation at room temperature

I am considering preserving the DNA of a family member who passed away. The funeral home offers a service called DNA Memories from a Canadian company (CG Labs). They have two options: Store the DNA in ...
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78 views

Why Cerebrospinal fluid is not heated for VDRL?

For performing VDRL of serum , we heat serum to inactivate complement proteins which may otherwise interfere , but why don't we do same for CSF even though it too has complement proteins in it? Is it ...
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1answer
77 views

How to watch a Zebrafish embryo in detail?

How much microscope zoom would I need to watch the development of a Zebrafish embryo in certain detail? Thanks guys!
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55 views

How is the role of ethylene in ripening demonstrated in biology (or agriculture) education?

I want to know what are some typical experiments performed in the course of biology education (or possibly in applied biology education e.g. agricutlure education) to prove to the students (or have ...
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1answer
74 views

When in Ampicillin degraded (gone) in liquid TB-media? Concerns about selectivity

Question: Specifically regarding Ampicillin; When growing cells in TB (terrific broth) for protein expression - when should I expect the ampicillin to be gone due to degradation by b-lactamases? (and ...
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1answer
69 views

How can enzymes be immobilised on glass?

I’m studying a hypothetical model for urease activity, which involves fluorescence measurement, hence the need for an optical window to which the enzyme urease is immobilised. From my previous ...
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34 views

Using distilled water on sterile swab for collecting microbes on solid surfaces

I was wondering if it is acceptable to use distilled water on a sterile swab for the collection of microbes on a solid surface. - in this case, Salmonella and Shigella.
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1answer
174 views

EnteroPluri test contamination

An EnteroPluri test has twelve different sectors. It seems odd to me that pulling the inoculating tip from the starting sector (citrate) all the way to the ending sector (glucose/gas) and then ...
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124 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 ...
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1answer
59 views

Can experiments in ELISA kits be monitored via both fluorometry and photometry?

In my bioanalytics course slides, the professor has written at one point that in a heterogenous immunoassay such as ELISA, we use fluorimetry to measure concentration of an analyte. In another slide ...
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43 views

Why is plasma glucose concentration not double that of whole blood?

It is known that the concentration of plasma glucose is 12% higher than that of whole blood. But since 45-50% of whole blood is red blood cells, shouldn't the plasma glucose be almost double — since ...
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35 views

Control for Bisulfite sequencing

I am wondering how can I control my converted DNA! I converted my DNA with bisulfite then I amplified the converted DNA with specific primers of BSP and purified my product and finally sequenced the ...
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Minimum number of cycles for effective qPCR quantification?

I have been having an apparent problem with gDNA contamination in my no-reverse transcriptase (NRT) controls by the appearance of fluorescence peaks in my qPCR data. I have/am trying multiple DNase ...
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1answer
704 views

Why shouldn't I clean microscope slides with a paper towel?

I've recently bought a microscope and been looking at a lot of bacteria and such, and have quickly run out of clean slides. I've seen videos and articles saying how you shouldn't clean microscope ...
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1answer
139 views

Method for preparation of LB agar plates with Ag+?

I need a method for preparing LB agar plates with 1 mM concentration of AgNO3. I can not seem to find a method online, does anyone know of one that I could use? Here are my main questions regarding ...