Questions tagged [protocol]
A predetermined methodology for carrying out an experiment.
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In enhanced chemiluminescence in western blots, will the horseradish peroxidase eventually get used up?
I am learning about enhanced chemiluminescence in Western blots. I have read online that in enhanced chemiluminescence that horseradish peroxidase (HRP) catalyses the oxidation of luminol to 3-...
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Why is it good practice to prepare western blot samples in pairs when you have two experimental groups?
I have done some Western blots where I have analysed the expression levels of a certain protein in brain homogenates from wild-type and knockout mice. When preparing brain homogenates, I was advised ...
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In phospho/pan analysis in Western blots, what is best way to normalise to an internal loading control?
I am analysing the expression of a protein kinase X that is a phosphoprotein through Western blots. I have labelled the membrane for both the phosphorylated form of the protein and also for the total ...
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When analysing phosphoproteins via Western blot, why is total protein level of the target protein recommended as an internal loading control?
I am analysing the expression of a protein kinase via Western blots, and it is a phosphoprotein. I have labelled my membrane with antibodies against the phosphorylated form of the protein (using ...
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Why do you need to calculate the lane normalisation factor when doing western blot data analysis?
I am learning about western blot data quantification from some online resources. I have read about methods to normalise the data.
I have seen in a number of resources, such as this handbook and this ...
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Is it possible to use PCR to test for Machado-Joseph disease?
Machado–Joseph disease (MJD) is a rare inherited neuromuscular disease that is caused by a mutation in the gene ATXN3. The protein encoded by this gene contains "CAG" repeats in the coding ...
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What is the best way from remove background signal from a band when doing Western blot image analysis?
I am doing image analysis of a Western Blot in Image J. I have calculated the total intensity of my protein bands of interest through outlining each band using the rectangle tool in ImageJ, and ...
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How can I convert plate reader measurements of Pichia pastoris OD to cells per ml?
I am performing experiments with the yeast Pichia pastoris where it will be beneficial to calculate product yield per cell. I plan to use a plate reader to measure OD600 of my cultures and then ...
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Pipetting: do human experimenters need liquid class information?
I've been working on a protocol standardization project where, among other things, we want protocols to be able to be run equivalently by both humans and robots.
Something that I've noticed in doing ...
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Effect of ethanol on TRizol RNA extraction
I added ethanol instead of chloroform to the cell suspension in Trizol. Can I still obtain my aqueous phase?
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Cellulase Sample Digestion Protocol?
Is there an effective way to use cellulase(s) and/or lignase(s) to remove unwanted plant debris from a sample?
I'm working with a series of fresh water grab samples for environmental assessment. The ...
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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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Flow cytometric counting of apoptotic adhering cells
I have been trying to measure rates of apoptosis in epithelial cells by marking apoptotic cells with annexin V conjugated with phycoerythrin (PE) and sorting using flow cytometry. Unfortunately, the ...
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How do you keep track of past/new lab protocols?
Does there exist a list grouped my subject (e.g. 'microscopy') for keeping track of what protocols have been designed and used?
Closest I could find is http://www.protocol-online.org/ but it's fairly ...
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What is the protocol for extracting Protease Onion?
I am Investigating the protease concentration in certain fruits and vegetables. I am unable to find the protocols for the extraction from onions. I want to purify my protein using the ammonium ...
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Assays for protein or lipid content of insect pupae
Does anyone have any recommendations (based on personal experience) for rapid, cheap, accurate assays for estimating protein & lipid content of individual insect (mosquito) pupae?
So far I've ...
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Reversal of cross-linking in ChIP-seq
Chromatin immunoprecipitation (ChIP) is a type of immunoprecipitation experimental technique used to investigate the interaction between proteins and DNA in the cell. A summary of the protocol for ...
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Storage DNA Condition
It is possible to store a digestion of cells with lysis buffer several days at -20 degrees and then proceed with the phenol chloroform isoamyl purification?
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What is the point of storing biological samples in Trizol/TriSURE before RNA extraction?
The Wikipedia article on Trizol says that it helps break the cells and maintain RNA integrity during homogenisation of samples. Does anybody know by how much Trizol improves RNA integrity compared to ...
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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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Should I use glycogen in isopropanol to precipitate RNA or does glycogen have harmful consequences on downstream protocols such as RNA seq?
I have heard that one can use glycogen in isopropanol to better visualise the RNA pellet after centrifugation. What are the advantages and disadvantages of adding glycogen in isopropanol for RNA ...
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Should I use RNAseZAP on my bench and pipettes before RNA extraction?
Should I spray RNAseZAP on my bench and pipettes before working with RNA? I am regularly using RNase at my bench and would like to work with RNA as well. Could traces of RNase on my equipment degrade ...
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Where is DNA during the process of RNA extraction using Trisure?
Where is DNA during the process of RNA extraction using Trisure described here? Given that DNA is also a nucleic acid, should I expect the isolated RNA to also contain DNA? However, Bioline claims RNA ...
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Why marker and control are loaded in the beginning or ending lane of the gel?
I have seen gel images in several literature. Almost in all, markers and controls are loaded in the extreme lanes (before 1st sample or after last sample) of the gel. I am just curious, is there any ...
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Tests for object size discrimination in mammals?
Are there common testing protocols for establishing whether an animal (mammals especially) can distinguish the size of objects? In particular, are there procedures or is there good literature on ways ...
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Difference between cytospin and metaphase spread protocol
I would like to know what exactly is the difference between cytospin and metaphase protocol. I'm a data scientist helping a medical lab and I receive images of cells with particular types of DNA. The ...
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How does one experimentally obtain a hemoglobin-oxygen dissociation curve?
I've always wanted to do comparative eco-physiology studies looking at Hemoglobin-Oxygen affinity of populations in different environments, but I can't find a protocol to experimentally measure the ...
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Can RNA be extracted from tissue suspended in formalin?
There are two tumor samples floating in a 10% formaldehyde solution (i.e formalin). Is there a protocol for RNA extraction under these circumstances? I am concerned that using the protocol for ...
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Genomic DNA resuspension after ethanol precipitation
I am having trouble resuspending purified human genomic DNA.
The genomic DNA is purified using phenol:chlorofom from ~ 1 mL of blood. After ethanol precipitation, I normally see a large pellet which ...
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Genomic DNA extraction protocol by salting out with NaCl, how to improve yield
In these papers
A rapid non-enzymatic method for the preparation of HMW DNA from blood for RFLP studies
A simple salting out procedure for extracting DNA from human nucleated cells
Modified salting-...
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Protocol to dilute DNA step ladder?
I need to run gels that are of not the "most" importance. So I do not want to waste alot of money on step ladder. How do I dilute DNA step ladder? Is there a general procedure, for I have tried ...
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Avoid Blood lyse color remain in DNA extraction
I tryed several times to extract DNA from freezed blood sample whit Qiagene kit but it gave very small amount of product due to good purity then I try extract manually by using CTAB PVP method and ...
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How to troubleshoot islet isolation
I have been getting very low yield from islet isolations from rats.
My procedure is as follows:
Inject the pancreas with 5mL Liberase (1 wunch unit/ml) in HBSS + HEPES through the CBD (decent ...
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BSA for trouble shooting failed PCRs?
I am using PCR to amplify a 1000bp region of the CytB mt gene. Some of the samples had not amplified and I wish to change the reaction components (or use additives like BSA) to attempt at getting ...
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Is NADPH unstable in UV light?
I am working on an enzyme activity assay using NADPH as a cofactor. The MSDS for NADPH Tetrasodium Salt, tells to store it in a place away from heat and light. Does this include UV light or just ...
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Explanation of protocol in DNA extraction experiment
In the Materials and Methods section of this paper on DNA extraction and analysis from hair I do not understand some parts of their protocol methods. In particular:
"The digested hair solution was ...
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What is the purpose of using two layers of gel in SDS- PAGE?
I just made a SDS-PAGE with a top layer of stacking gel and a bottom layer of separating gel with different pH values of 0.5M Tris-HCl. The stacking was 6.8 and the separating gel was 8.8. What about ...
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Time required for RNA precipitation in ethanol
I precipitated bacterial RNA during 1 hour at -80ºC after depleting rRNA with Ribo-Zero kit.
Does more time lead to better results?
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Can bacterial RNA be degraded at -80⁰C in a SDS solution?
I would like to know if bacterial RNA in a 1.5 mL 2%SDS solution can be degraded.
Comes from a pellet of pure liquid culture.
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Change solvent of bacterial solution
I have to perform an RNA extraction that should start with 900 uL of 2%SDS bacterial solution. The problem is that the sample is in 900 uL of PBS.
I thought about centrifugation of the sample at 5ºC,...
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Estimating RPM to RCF in Methods from Older Papers
I'm attempting to replicate a cell biology method from a 1958 Laboratory Investigation paper. The protocol is for the isolation of an extracellular matrix protein, and a key step is a centrifugation ...
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What's up with all the vague protocols? [closed]
I have lost count of how many protocols I've seen, including those supposed to be professionally written (such as manuals that come with kits from well known brands, or methods sections of papers in ...
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How can I label cryotubes in a way that eliminates the problem of legible hand-writing?
My lab stores biological material (tissue, cells, plasma, serum) in a -130 C, liquid nitrogen freezer. The cryotubes that we use to store a samples are labelled by hand which frequently creates ...
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Can I use grayscale images when working with ImageJ?
I am using ImageJ to analyze Western Blots. I have scanned films in as grayscale images because this is how we did it in my old lab. People in my current lab are not satisfied with that explanation ...
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Appropriate Buffer for electerophoresis of DNA & Protein TBE or TAE?
Which buffer is best for DNA Electrophoresis and which is best for Protein to be have a sharp bond?
Considering a higher electrical conductivity compared to TAE & TBE and the generation of less ...
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can a bacterial protease inhibitor cocktail be used for Western Blotting involving HUVEC cells and HT-29 adenocarcinoma cells?
Dear fellow biochemists,
I need some advice on Western Blotting, more specifically the use of certain protease inhibitors with the RIPA cell lysis buffer and protease inhibitor cocktail. A Millipore ...
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Why extract DNA from certain white blood cells instead of whole blood?
In my lab, human DNA is extracted from whole-blood samples.
I don't actually do the extractions and I am not familiar with the specific protocol but I understand that platelets and red blood cells ...
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Machine learning for light microscopy -- problems to solve?
I would like solve some biological problems that would improve the state-of-the-art of biology or bioinformatics. In particular, I want to apply machine learning on light microscopic images. The ...
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What is "bacto" peptone?
Standard recipes for yeast medium often include "bacto-peptone". Is this the same as bacteriological peptone? Is there an authoritative source that spells it out?
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Is a sequential double transformation acceptable?
Standard protocol states having two compatible vectors being transformed simultaneously during the same procedure. I've come across a situation in which transforming one vector, obtaining results, and ...
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