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Questions tagged [protocol]

A predetermined methodology for carrying out an experiment.

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RNA isolation problem in Drosophila larvae

I am trying to isolate total RNA from Drosophila larvae using TRIzol method. I am using 30-40 larvae for the same. Is it an appropriate number for RNA isolation? I got 260/280 ratio as 1.8 and 260/...
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1answer
42 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 ...
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15 views

How to dissociate a viral particle into constituent proteins?

Is it possible to take purified a virus particle and dissociate it into a mixture of its constituent proteins (and nucleic acids)? I am wondering if it would be possible to disassemble the particle ...
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1answer
178 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 ...
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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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13 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 ...
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1answer
46 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 ...
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1answer
30 views

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? Thanks.
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44 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 ...
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124 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 ...
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35 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 ...
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18 views

Incomprehensible Bioanalyzer profile

I have extracted RNA from about 4000 C. elegans worms today, using my Trisure and phase-lock gel protocol. The ratios at the Nanodrop are excellent, indicating that there is not protein or salt ...
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1answer
131 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 ...
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1answer
63 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 ...
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1answer
34 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 ...
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10 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 ...
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50 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 ...
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56 views

dissolve agar gel to retrieve whole plant

We grow Arabidopsis thaliana on agar plates. In order to fix them for later experiments, we currently collect the plants one by one out of the agar plates to transfer them in a PBS solution. With many ...
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84 views

small pellet size after protein production in E. coli

I expressed a protein using 0.5 mM IPTG in E. coli (BL21 cell line straight from a glycerol stock was used as starter culture, ampicillin as antibiotic at 100 mg/L) but the cell pellets were very ...
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1answer
100 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 ...
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1answer
781 views

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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1answer
1k 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 ...
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652 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-...
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1answer
876 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 ...
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41 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 ...
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61 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 ...
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0answers
76 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 ...
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1answer
68 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 ...
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1answer
825 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 ...
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18k 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 ...
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2answers
2k 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?
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1answer
216 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. Thanks
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2answers
101 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,...
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1answer
75 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 ...
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98 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 ...
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2k 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 ...
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1answer
622 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 ...
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4answers
2k 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 ...
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1answer
171 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 ...
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1answer
11k 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 ...
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4answers
771 views

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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1answer
2k views

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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597 views

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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1answer
428 views

Function of heparin and dextran sulfate for removing proteins

From this article : The reaction was terminated and the histones, and most nonhistones, were removed by adding the nuclease-treated chromosomes to a solution containing dextran sulfate (2 mg/ml)...
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1answer
116 views

Changing time and rpm of centifuge

Inspired by this question, I want to ask a question about centrifugation. Suppose a protocol says : 10 min at 2500 rpm . Can we instead centifuge for 5 min at 5000 rpm or 20 min at 1250 rpm ?
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1answer
117 views

Concentration of DNA by isopropanol

I have read that DNA can be concentrated by addition of isopropanol. What does "concentrated" mean? What does isopropanol do on a molecular level to concentrate DNA?
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1answer
2k views

circularizing DNA molecules?

I have been reading about next-generation sequencing technologies that can sequence long reads. Even though the origin of my question is sequencing technologies, the question I am asking is about the ...
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1answer
5k views

How to avoid air bubbles while pipetting?

I get air bubbles while pipetting small volumes. How can I avoid them ?
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1answer
661 views

How do scientists create specific mutations?

Suppose I want to create a mutant like Antennapaedia how will I go about accomplishing it ? I know that radiation and certain chemicals are mutagenic. So do scientists subject animals to such ...
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1answer
79 views

Why prefer $MgSO_4$ over $MgCl_2$ in recipies [closed]

Many molecular biology recipes use MgSO4 (and not MgCl2). Is there indeed a preference? If so, why?