Questions tagged [lab-techniques]

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

72 questions with no upvoted or accepted answers
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6
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54 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/...
6
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1answer
175 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 ...
5
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1answer
143 views

Optimization of Signal Peptides

We are looking at modifying the signal peptide of a receptor in a common immortalized human cell line. The cell line already expresses an unusually high amount of the protein, but much of it is not ...
4
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0answers
2k views

How much salt [NaCl] is too much in DNA precipitation?

In DNA extractions, how much is too much salt in a CTAB extraction buffer? Protocols hover around 2.5 molar; if you go over this (e.g. 25 molar), will you saturate your solution, and precipitate the ...
4
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0answers
53 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 ...
3
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0answers
22 views

How does a heat-ratio sap flow sensor work and how invasive is it?

The Gizmodo article Undead Tree Stump Is Being Kept Alive by Neighboring Trees says: Leuzinger and Bader stumbled upon the stump while out for a hike. The woody stub caught their eye because callus ...
3
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0answers
44 views

Can anyone expolain this? Cell culture vials have strange hair-like condensation patterns

What is the hair-like polymer that condenses on the caps of the plastic cell vial? Inside vial are cells in a mixture of FBS and DMSO. The vials were frozen slowly in a special isopropanol-filled ...
3
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0answers
88 views

Travelling with tissue samples in RNAlater

I've been in Brazil collecting some samples (ants) and need to travel back to to the UK with - I've got their brains stored in RNAlater, which have been in the freezer at -4C for a bit less than a ...
3
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0answers
35 views

How do I test if microbes have survived after dehydration?

I have a solution containing various bacteria and fungi. My aim is to place solution on filter paper, and wait until it dries. I then wish to test if the organisms have survived, either on the dried ...
3
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0answers
605 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, ...
3
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0answers
256 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, ...
3
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0answers
459 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 ...
2
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0answers
9 views

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, ...
2
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0answers
127 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 ...
2
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0answers
42 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 ...
2
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0answers
18 views

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 ...
2
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0answers
85 views

Would I expect salt precipitate on fibres of DNA in a NaCl water solution?

I just recently conducted an experiment in my biochemistry class where we had to add DNA to distilled water, isotonic saline and 2.5M NaCl. I noticed different physical aspects depending on the ...
2
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0answers
157 views

At what g force do bacteria start to pellet down a tube?

I am working with C. elegans and bacteria and I want to get rid of the bacteria they eat by centrifuging the worms without centrifuging the bacteria. I am using a g-force of 600 and the bacteria ...
2
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0answers
82 views

What happens during fixation of Nucleic acids on nylon membrane

In Southern blotting technique, fixation of DNA to nylon membrane is a step prior to hybridisation with probe. It is done by baking at 80ºC and according to this cross linking between DNA and nylon ...
2
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0answers
37 views

How urination prior to blood test may give error?

Source: Textbook of Practical Physiology, 4th Edition according to it urination within 30 minutes is a precollection factor that may alter the results of blood test. I am unable to comprehend how ...
2
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0answers
13 views

Propagating errors for percentage reduction

Need a bit of help for this particular problem. I have three averages collected from three genotypes (A, B, and C) and their associated SD (a, b, and c). A is my control. I have been asked to ...
2
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0answers
35 views

Nucleic acid extractions

Is it fine to leave pulverized plant tissue in CTAB buffer at -80 degrees over night? will the buffer remain effective to generate good quality nucleic acids?
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64 views

Where am I going wrong in a Reactive Oxygen Species Assay done in N9 cells?

I have followed the modified protocol that was given to me by my senior (who was using for his RAW 264.7 cells - Reference article: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3308605/ ) for ...
2
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0answers
67 views

PCR limits of detection calculate method

Is any standard method for use to gain PCR sensivity or limit of detection? my mean is not for RT-PCR or Real-Time or any other kind and just simple PCR. After making PCR product free of dNTP and ...
2
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0answers
74 views

Do users of CRISPR/Cas iterate or parallelize to try multiple guide sequences?

I've read about on-target efficiency and off-target effects in use of CRISPR/Cas9, and about tools that suggest good guide sequences. I am wondering: how many guide sequences do typical CRISPR users ...
2
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0answers
38 views

Stable isotope sample preparation: Bone Collagen

I am performing research in which I need to extract bone collagen from the foot bones of American Beaver (Castor canadensis). I have read through the literature fairly thoroughly on both the ...
2
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0answers
38 views

How would one identify cellular transcription factors associated with a viral protein in a treated cell line?

I've been working as the computer guy for a microbiology lab for the past few months. I've always been interested in bench work, but my wet lab experience is rather limited and thus so is my ...
2
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0answers
196 views

Why do I get such a strong background in a detection of DIG-labeled DNA (Southern Blot)?

I have done a Southern Blot Analysis of DNAmt transferred to nylon membranes. The DNA was firstly loaded on a 2% agarose gel. An immunoassay was done to detect the bands using Anti-Digoxigenin ...
2
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0answers
68 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 ...
2
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0answers
500 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 ...
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0answers
13 views

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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0answers
51 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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79 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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0answers
56 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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0answers
36 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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0answers
133 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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0answers
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 ...
1
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0answers
163 views

What are the optimal conditions for storing frozen glycerol stocks of bacteria?

The common method of storing bacteria long-term is to collect saturated liquid culture, such as LB, and mix it with sterile glycerol so as to obtain 10-30% final glycerol concentration. The resulting ...
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0answers
50 views

What are these colonies on my Bacillus pumilus lawn?

I am growing Bacillus pumilus and I have plated some on agar plates. Now I can see some colonies on top of the lawn. Does anybody know what they could be? A contaminant? Bacillus pumilus? I have ...
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0answers
25 views

How to convert a test tube assay to microplate assay?

I am trying to measure Ascorbic acid levels in tissue using Omaye et al protocol (Methods in Enzymology, Volume 62, 1979, Pages 3-11). This method is based on reduction of ferric chloride and ...
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0answers
31 views

What is known and what can be measured of biofilms?

Context for the question I am a mathematics researcher working on the mathematics of imaging and parameter estimation (inverse problems). I found an interesting diffusion equation that models the ...
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0answers
168 views

How to keep Paraffin Sections from falling apart?

I've been trying to make 5 uM sections of injured mouse spinal cords and stain the tissue with Luxol Fast Blue and H&E. At first I was removing the bones from around the spinal cord, but the ...
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0answers
94 views

What are preparation mistakes that could make red blood cells appear crenate in isotonic solution?

Thank you for your time. I separated red blood cells from a whole blood sample purchased from a blood bank. In isotonic solution, the red cells appear crenate. I would like to know if there is ...
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0answers
66 views

Plasmid transformation yields only empty vectors

I have to electroporate pMAD cloned with an insert of 7.4 kb in my S. aureus isolate. When I electroporated in RN4220 I had no problems but when I put it in my isolate I just recover few colonies and ...
1
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0answers
209 views

Why are my assay absorbance values very low?

I am culturing SH-SY5Y cells in 96-well plates at a seeding density of $1.2 \times 10^5$ cells/mL. I differentiate the cells with retinoic acid for 6 days before giving them treatments. The cells look ...
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0answers
34 views

How could you measure the rate of light reactivity/ photosynthesis of a cell in a lab setting?

For a lab I must take Volvox protist cells and expose them to specific wavelengths of light. How would I know they're even reacting? The assignment I've been given does not explain what I should even ...
1
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0answers
37 views

How did researchers find out that it was only the maternal chromosome that underwent deletion in Angelman syndrome?

It is known that in Angelman syndrome, the chromosome 15 undergoes deletion in the maternal chromosome while in Prader-Willi it's in the paternal. I understand it can be detected by FISH or other ...
1
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0answers
274 views

DAPI staining showing unknown artefacts?

Dear fellow researchers, I have been facing some problem with staining nucleus with DAPI. I use the standard protocol for fixation 4%PFA+0.1% TX-100+2%BSA. I use 0.2ug/ml conc. of DAPI for 5 minutes....
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0answers
45 views

What is the typical amount of full length clones in a cDNA library?

For a Yeast Two Hybrid project we need to make a cDNA library from a single celled organism. We used the Cloneminer II kit from Thermo. We followed the manual and performed all the quality control ...
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0answers
107 views

Effective Mycoplasma Elimination from Primary Human Cultures

Obviously the best way to avoid mycoplasma contamination is to avoid it in the first place. In our case, however, it is not possible to avoid. We are culturing viruses and tissues out of human nasal ...