Questions tagged [lab-techniques]
Questions relating to protocols, procedures, and good practice when using laboratory equipment.
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Help with preparing Congo Red TSA
So I want to make Congo Red TSA. My instructor gave some hints but I still have some questions:
I start by making two stock solutions: 20 mg/ml of congo red and coomassie in two different falcon ...
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1
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Is it possible to produce a tangible mass of DNA/RNA using a PCR reaction?
A Polymerase Chain Reaction (PCR) is a method of amplification which enables one to produce many copies of a certain DNA/RNA strand for many applications (as described in the link above).
I have ...
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Purpose of dilution streaking or streak purification
In dilution streaking a drop of liquid culture is collected with an inoculating loop and then streaked across the agar plate surface. At the end of the streaking the number of organisms clinging to ...
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What for a Kinase Assay? [closed]
I am wondering why to use a kinase assay, since we can extract the proteins from cells and then do a Western with the specific antibodies we want to use.
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Voltage sensitive dyes technique: What is the underlying measure?
I just discovered voltage sensitive dyes technique: first of all what imaging techniques do we use? And I have seen that figures are labeled with ΔF/F0, what does it stands for?
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Optimizing Gel Electrophoresis: Ampere, Volts and Buffer concentrations
I am a master student in biochemistry, and I have used gel electrophoresis many times before. What I want to know is how one should adjust the mA (mAmpere) compared to the voltage and the buffer one ...
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3
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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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Career progression through biosafety levels?
Does a career in infectious disease typically progress through biosafety levels, or do people select one and specialize their training in procedures specific to those hazards?
That is, say Dr. ...
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Why does my anti-ubiquitin antibody visualization not work on my PAGE gel?
I am using 2D gel electrophoresis to visualize polyubiquitinated proteins. However, while I can see actin and heat shock protein using when appropriate antibodies, I cannot visualize them using anti-...
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Does methanol fixation deform cultured cells?
I use methanol fixation (@ -20⁰C for 10 minutes) when performing immunofluorescence assays on cultured cells. Generally speaking, this results in very good antibody staining. However, the cells tend ...
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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?
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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 former,...
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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 ...
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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 ...
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microscopic slide cleaning and maintenance
we do lot of gram staining and some spore staining. we normally wash our slides with detergent then rinse in D/w, dry it and store it. But on reusing this slide we have to flame it and also clean it ...
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Common mistakes when sequencing?
I've sequenced two genes of 15 dogs for SNP analysis. the chromatograms of one gene looked perfect but the ones of the second gene didn't look as good. To be precise, the chromatograms of one exon ...
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Effect of ethanol and detergent on cell membranes problem
I am currently writing my biology report for an experiment I did on how ethanol and detergent affect the cell membrane. For my ethanol experiment, all went as expected. However, for my detergent ...
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Where does the inverse seconds unit come from in the association constant?
I'm working to determine Kd(s) kinetically by generating association and disassociation curves. Kon (association constant, or on-rate) is in inverse seconds multiplied by inverse molar. I get that ...
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Extending a small fragment of DNA
Is there a way to extend a small fragment of DNA, say 150 bp, by making copies of itself and attaching each copy of that small fragment to the end of that 150 bp sequence?
For example, I want a 1 ...
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Workspace preparation and cleanup for DNA work [duplicate]
What steps should be taken in a molecular lab environment to help ensure that DNA samples/stocks are not contaminated, or contaminate other objects in the lab?
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How long can a human neuron live outside the body in a controlled environment?
Have there been any experiments that have kept neurons alive (stationary), without preserving methods such as freezing? If yes, then how long were the cells kept alive for?
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How does a TOPflash/FOPflash assay work to detect beta-catenin protein expression?
I am reading an article (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3596711/) wherein a TOPflash/FOPflash assay is used to detect beta-catenin protein levels in a COS-7 cell line. I can't find a good ...
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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 ...
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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:
...
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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 ...
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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 ...
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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:
...
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Does glycerol in E.coli culture media somehow inhibit the lac-operon?
I have have been taught that one should induce protein expression with IPTG at an OD of about 1.0 - 2.0 when E.coli grows it TB media (terrific broth). As a reference point, one typically induces ...
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RNA product storage in isopropyl alcohol
I have an issue. I isolated RNA from cortex a few days ago. For isolation of RNA i used this procedure:
I homogenizised my sample (mouse cortex) with Trizol. Then to the sample add chloroform, shooke ...
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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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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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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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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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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 ...
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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 ...
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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 ...
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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 ...
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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 ...
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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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plausible science for why an enzyme breaks down a substrate better at 4 degrees rather than 20 degrees [closed]
An enzyme was found to have an optimal temperature of 20 degrees. The same enzyme in sheep liver extract was found to have an optimal temperature of 4 degrees. What are some of the factors that can ...
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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 ...
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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 ...
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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 ...
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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 ...
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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 ...
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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 ...
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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 ...
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How does air-liquid interface (ALI) culture work?
For studying diseases, such as chronic obstructive pulmonary disease (COPD), cells are often grown in air liquid interface.
I understand that the common way of establishing this these days is to grow ...
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How to attract Cyanobacteria?
I would like to attract cyanobacteria in one spot on an object (e.g. cloth,) instead of having it swim in the media. my current method is to pour it on the object in a beaker and wait for some of them ...
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Why spin live fruit flies in centrifuge?
I was reading this article about the evolution of monarch butterflies’ resistance to cardiac glycosides: These Butterflies Evolved to Eat Poison. How Could That Have Happened?
And this passage made me ...
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