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Questions tagged [protocol]

A predetermined methodology for carrying out an experiment.

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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 ...
Real Fly's user avatar
2 votes
1 answer
38 views

Improving contrast between dot and paper in dot blot

Currently using dot blot to attempt to determine if a serum contains antibodies for some reagents I am testing. I pipetted samples as 10-5ul dots on whatman paper. Incubation steps were for 1 hour ...
Thomas Hunt's user avatar
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Is there a way to isolate bacteria from a bacteria/phage plaque in order to PCR bacteria only?

I am writing a paper for a class that involves a mock grant proposal. I am looking at an experiment which requires sequencing of host DNA from a virus/host model, and am having a hard time finding a ...
Erin Marble's user avatar
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1 answer
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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 ...
ceno980's user avatar
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2 votes
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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 ...
ceno980's user avatar
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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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1 vote
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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 ...
ceno980's user avatar
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3 votes
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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 ...
Nova's user avatar
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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 ...
ceno980's user avatar
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6 votes
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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 ...
Noah Sprent's user avatar
21 votes
4 answers
2k views

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 ...
jakebeal's user avatar
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4 votes
1 answer
187 views

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?
polonio210's user avatar
1 vote
0 answers
54 views

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 ...
L84Dnr's user avatar
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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. ...
Carmen Sandoval's user avatar
3 votes
1 answer
183 views

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 ...
BelowAverageIntelligence's user avatar
4 votes
1 answer
223 views

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 ...
Adam Ashwal's user avatar
2 votes
0 answers
121 views

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 ...
TescoExpress's user avatar
2 votes
0 answers
23 views

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 ...
arboviral's user avatar
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4 votes
1 answer
2k views

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 ...
Anu014's user avatar
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1 answer
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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?
Serra's user avatar
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1 vote
0 answers
75 views

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 ...
charlesdarwin's user avatar
2 votes
0 answers
251 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 ...
charlesdarwin's user avatar
1 vote
0 answers
76 views

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 ...
charlesdarwin's user avatar
1 vote
1 answer
409 views

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 ...
charlesdarwin's user avatar
1 vote
1 answer
250 views

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 ...
charlesdarwin's user avatar
1 vote
1 answer
52 views

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 ...
RKK's user avatar
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1 vote
0 answers
11 views

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 ...
orome's user avatar
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2 votes
0 answers
86 views

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 ...
user avatar
2 votes
1 answer
182 views

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 ...
Kara's user avatar
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1 vote
1 answer
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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 ...
Arthur Miller's user avatar
2 votes
1 answer
2k views

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 ...
Green's user avatar
  • 273
1 vote
0 answers
975 views

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-...
Green's user avatar
  • 273
0 votes
1 answer
2k views

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 ...
Ro Siv's user avatar
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0 votes
0 answers
58 views

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 ...
M007's user avatar
  • 588
1 vote
0 answers
78 views

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 ...
Jesper Madsen's user avatar
2 votes
0 answers
89 views

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 ...
Anurag Mishra's user avatar
2 votes
1 answer
73 views

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 ...
wswr's user avatar
  • 21
2 votes
1 answer
987 views

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 ...
Ro Siv's user avatar
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11 votes
2 answers
39k views

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 ...
Stacked's user avatar
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5 votes
2 answers
3k views

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?
biotech's user avatar
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5 votes
1 answer
234 views

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.
biotech's user avatar
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3 votes
2 answers
107 views

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,...
biotech's user avatar
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3 votes
1 answer
93 views

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 ...
Chastain's user avatar
  • 311
4 votes
1 answer
130 views

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 ...
Superbest's user avatar
  • 4,520
11 votes
4 answers
3k views

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 ...
Slavatron's user avatar
  • 1,010
2 votes
1 answer
1k views

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 ...
Slavatron's user avatar
  • 1,010
5 votes
4 answers
3k views

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 ...
M007's user avatar
  • 588
2 votes
1 answer
235 views

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 ...
Robert Weinberg's user avatar
7 votes
1 answer
13k views

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 ...
Slavatron's user avatar
  • 1,010