Tagged Questions

A predetermined methodology for carrying out an experiment.

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9
votes
3answers
72 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 ...
1
vote
1answer
39 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 ...
5
votes
4answers
141 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 ...
1
vote
1answer
31 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 ...
6
votes
1answer
127 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 ...
13
votes
4answers
273 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 ...
3
votes
1answer
77 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?
5
votes
1answer
70 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 ...
3
votes
1answer
49 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 ...
3
votes
1answer
25 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 ?
1
vote
1answer
32 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?
1
vote
1answer
112 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 ...
5
votes
1answer
542 views

How to avoid air bubbles while pipetting?

I get air bubbles while pipetting small volumes. How can I avoid them ?
6
votes
1answer
180 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 ...
0
votes
1answer
59 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?
1
vote
1answer
76 views

Agglutination test using antibodies

Agglutination test Latex agglutination using bound antigens : by coating soluble (non - particulate ) antigens on to microscopic latex spheres, their reaction with a particular antibody can be ...
2
votes
1answer
41 views

Allergic rhinitis vaccine

Note : Any answer to this question will not be (and should not be) taken as medical advice. One of my friends has allergic rhinitis and has been prescribed an oral vaccine. He is allergic to 3 ...
1
vote
1answer
41 views

reason for “not for diagnostic use” advice in protocols

I read "not for diagnostic use" in every protocol i saw. why is it so? which differences have the kits and material that are used for diagnostic purposes?
2
votes
3answers
130 views

Bacterial cell lysis buffer used in proteomics procedures

What kinds of detergent-free bacterial lysis buffers exist? The proteins we're extracting will be later analyzed by LC-MS/MS, and we're looking for a lysis buffer that won't interfere with this ...
3
votes
1answer
486 views

Problem with bacterial transformation with electroporation

I have a problem with a bacterial transformation of a yeast gene that I can not solve. I isolated yeast DNA and did a PCR to get my product. I am using pCGCUm vector with a GFP construct. I digest ...
4
votes
1answer
848 views

Basic step by step methods for PCR & Gel electrophoresis class

I'm teaching a class how to do PCR & gel electrophoresis soon and would like it if you could check through my basic step by step instructions - it's a while since I've done one. Is there anything ...
1
vote
2answers
59 views

Protocol for measuring stamina of arthritic mice

I am going to make some mice ill with arthritis. The medicine which I am testing as control is known to cause extreme fatigue. I think I've improved it. I'd like to humanly measure their stamina in ...
4
votes
2answers
610 views

How does formaldehyde/PBS or methanol fixation of cells affect lysosomal pH?

The question is fairly simple - does formaldehyde or methanol fixation in preparation for immunocytochemistry/immunofluorescent staining affect the pH of the lysosomes? Some background: I'm trying to ...
5
votes
2answers
348 views

High Current (Speed) Transfer Buffer Recipe

Does anyone know an effective buffer mix to use for high current Western transfers? We are successfully using the vendor's premixed buffer to transfer a wide range of protein sizes to PVDF membranes ...
1
vote
0answers
737 views

Low RNA yield, low 260/230 ratio

I extracted total RNA from animal tissue using the Qiagen RNeasy kit, however my RNA yield was extremely low and the 260/230 ratio was around 0.3. This is the protocol I followed: The animal ...
1
vote
0answers
54 views

Removing bacterial contaminants from resin

We have a few Strep-tactin columns that had some growth in them and we would like to regenerate the columns back since the resin is quite expensive. Basic goal: remove the brown stuff. So far, I've ...
1
vote
0answers
137 views

Ligase Chain Reaction: Determining the annealing temperature for gene synthesis

Using the tool Gene2Oligo I have a set of DNA oligonucleotides to synthesize a gene using ligase chain reaction (LCR). The average melting temperature is 72 degrees Celsius. I am going to use a the ...
1
vote
1answer
40 views

In vitro enzyme production

I need to express a protein in vitro but I don't know where to start. I will likely do a T7 transcription protocol but for translation I am not sure what to do. Are there any good kits?
2
votes
0answers
49 views

How to use mechanical microstrainer to extract tissue proteins from human?

Background: There are many methods to extract proteins form human tissues out there. The majority of them use an extraction buffer containing variable concentrations of detergents and protease ...
1
vote
0answers
19 views

activate a drug using deoxycytidine kinase in vitro

I need to activate a drug similar to the way it is activated in the cell. My set of molecules called Nucleoside analog reverse-transcriptase inhibitors (NARTIs or NRTIs) need three phosphate groups to ...
2
votes
1answer
391 views

Using DTNP to find free thiol groups on a protein

I've been tasked with using DTNB to find the number of thiol groups on a molecule of Bovine Serum albumin (BSA). After measuring the absorbance, finding the concentration of TNB and calculating the ...
1
vote
2answers
329 views

PEG-silane treatment: why incubate for 18 hours at 60 degrees Celsius?

I am conducting a biochemistry-related experiment and I have been unable to understand a step which is commonly performed. My aim in this step is to apply a PEG (Polyethylene glycol) silane layer. ...
4
votes
1answer
201 views

Can I heat Trizol?

I wonder if I can heat Trizol reagent for 30 min 65C. The goal is to disrupt protein-RNA complex while inhibiting nucleases. (I can't use RNasin cause it's inactivated in 65C, and can't use RVC cause ...
4
votes
2answers
95 views

Low temperature PCR

We're trying to do emulsion PCR using HA-coated polystyrene beads and we're noticing that the beads are seeing drastic issues with thermal degradation above 90C. As PCR has an unfortunate requirement ...
3
votes
1answer
105 views

Cell growth conditions for preparing electrocompetent cells

Usually the protocol for preparing electrocompetent E. coli cells calls for growing the cells at 37deg and 225rpms until they reach OD of 0.3. I was wondering, is there any reason they should grow at ...
10
votes
4answers
800 views

Why is it sometimes difficult to resuspend E. coli in P1?

It's a curiosity question. When I'm doing minipreps after pelleting the bacteria sometimes it's very easy to resuspend them in P1 (Qiagen kit), but sometimes they form a rubbery clump that is very ...
3
votes
1answer
554 views

Open protocol for Ligase Independent Cloning

Ligase Independent Cloning is a protocol that allow an insert to be integrated into a vector without ligation. It uses T4 DNA polymerase with only ATP to first chew back from blunt ends to create ...
4
votes
2answers
710 views

Why is proteinase K digestion performed at 50 °C?

Many DNA isolation protocols use a Proteinase K digestion to remove proteins. This is often performed at 50 °C. Why is this?
2
votes
0answers
95 views

Has anybody used Evrogen's DSN-normalization protocol for cDNA normalization? [closed]

I found the link to a commercial product by Evrogen to normalise cDNA samples for gene discovery projects here: http://www.evrogen.com/technologies/normalization.shtml The most up-to-date reference ...
13
votes
1answer
194 views

How straightforward is in vitro compartmentalization?

in vitro compartmentalization (IVC) is one of those technologies that everyone knows about, talks about, but never actual does due to the rather technical difficulties in setting the system up. I was ...
11
votes
2answers
172 views

protocol for pulldown of DNA breakpoints?

Is there any method to do pulldown enrichment of DNA breakpoints from a cell? I have found this paper reporting a method to enrich for the DNA single-strand breakpoints from meiotic recombination ...
5
votes
2answers
804 views

How to set up a slow cooling on an AB Veriti thermal cycler?

I want to incubate my sample at 50ºC and then slowly cool it down to 4ºC at a rate of 0.1ºC/s. I am using AB Veriti thermal cycler. Does anyone know how to set up the rate of cooling? It has to do ...