Questions relating to protocols, procedures, and good practice when using laboratory equipment.

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4
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0answers
39 views

Procedure for doing western blot [closed]

I am writing a step by step guide for doing a western blot for a class. It is intended for any one with basic Biology lab skills. I am hoping people will review my draft and give feedback on how to ...
2
votes
1answer
514 views

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 ...
3
votes
1answer
87 views

Safety of using industrial water for hand washing

Industrial water taps are present in most biology laboratories I have been too. There are accompanied by a sign stating "do not drink". So obviously it is not a good idea to drink it. However I ...
3
votes
1answer
90 views

Well to well variation in thermal cycler fluorescence?

We have an old BioRad ICycler Thermal Cycler with MyIQ single color fluorescent detector. While it's meant for RT-PCR, we've been using it for melt curves and binding assays for different types of RNA ...
2
votes
3answers
2k views

Long term storage of agarose-ethidium bromide gels that have already been cast

I have come up with what I thought was a clever idea: Store the agarose gels I pour, and only cut as many lanes as I need to run later, minimizing wasted agarose (and wasted effort/time) when I need ...
3
votes
1answer
149 views

How can I purify RNA after gel electrophoresis to remove residual acrylamide?

I sometimes use denaturing gel electrophoresis on a preparative scale to purify RNA produced by in vitro transcription. The major issue with this is that the sample after extraction from the gel still ...
6
votes
3answers
7k views

Should I dilute DNA with water or elution buffer?

I've extracted DNA using a kit and final step is eluted with buffer. If I must dilute my DNA samples, do I use the same elution buffer or can I use milliq water?
1
vote
0answers
182 views

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 ...
4
votes
1answer
11k views

NaCl role in CTAB - DNA complex in DNA extraction

I have a question about the role of NaCl in the DNA extraction process. So for NaCl concentrations under 0.5M, CTAB and DNA molecules can create complexes. In those concentrations, proteins and other ...
4
votes
1answer
68 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 ...
1
vote
2answers
3k views

Purifying a linear plasmid after restriction digest?

I expressed a yeast vector in E.coli and purified about 13µg of it. I then linearized it using a restriction enzyme, and attempted to gel purify it. I attempted this twice. The gel showed a clear ...
3
votes
1answer
3k views

How to prepare sample for SDS PAGE?

I usually take my protein sample 0.8ml and sample buffer(2X) 0.2ml for my sample preparation in SDS PAGE. Am I using correct proportion? my protein sample concentration is 4.1mg/ml. What is the ...
6
votes
2answers
465 views

poor RNA quality from zebrafish embryos

Does anyone routinely do RNA isolation from zebrafish embryos? I have embryos from different stages but all below 24hpf. This is the protocol I follow: Take 10-20 embryos Wash once with milliQ ...
1
vote
1answer
192 views

How to separate peptide on SDS PAGE?

I want to separate 1600 Da peptide on SDS PAGE, on 20% GEL I didn't get any band, How can I separate on PAGE? Can I use 25% SDS PAGE?
5
votes
2answers
3k views

Is wiping with RNAse Zap enough to destroy RNAse activity?

From the RNAseZap MSDS, it is an SDS at some unknown concentration, maybe with some NaOH? Some other links suggest there is some NaOH as well. The Ambion site states that RNAseZap destroys RNAse ...
3
votes
1answer
271 views

Ideal lab glassware cleaner for molecular biologist

I'm using Alconox powder detergent to clean my laboratory glassware, the powder is a pain to store near the sink and to use. Would like to make a concentrated liquid to use in its stead. I have ...
6
votes
2answers
2k views

Alternatives to trypsin for cell detachment?

I have ran out of trypsin and need to passage my cells (immortalized chondrocytes, C28/I2) today or tomorrow. I have been out of town and forgot to order more trypsin. I was wondering if there are ...
5
votes
2answers
635 views

How do you visualize RNA on a gel?

I have run an in vitro transcription reaction and produced some RNA of a single species and definite size. I would like to visualize it to check if the reaction worked. Can I do this on an agarose ...
9
votes
1answer
477 views

How do I put on a labcoat?

Let's assume I am wearing a labcoat for two reasons: Prevent the various bacteria, proteins, skin cells and substances on my clothes and my skin from contaminating my experiments. Prevent various ...
4
votes
1answer
51 views

Effect of Ethanol on Brain Volume Measurements

I want to compare the brain sizes of two populations of fish. However, all the samples I have were fixed in 95% ethanol. As far as I know, 4% formalin is the normal fixative for soft tissues. Can I ...
4
votes
2answers
122 views

Short, concise, practical manual for doing experimental biology

I am am physical scientist working in biology, and have recently started doing experiments. I would like a manual akin to "Numerical Recipes", but for the lab: straight forward, easy instructions on ...
2
votes
1answer
9k views

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 ...
2
votes
0answers
212 views

Origin of the 260/280 ratio?

This is not a duplicate of all the other 260/280 ratio questions, I already know that DNA is supposed to be 1.8 and RNA is supposed to be 2.0. However, this might be more appropriate for chemistry, ...
3
votes
2answers
81 views

What is an Ihh-/- mouse?

This one is too basic question: I just came across $Ihh^{-/-}\ $ mouse. Is that means this mouse devoid of that gene Ihh. What is this sign called and are there other such representations?
4
votes
1answer
2k views

DpnI over-digestion

We have a long protocol that we are optimizing that includes DpnI digestion of a PCR product (to remove any of the template DNA if it's methylated, and while we're not certain in the blind tests, ...
1
vote
1answer
74 views

What is an effective method for putting on blue rubber butyl stoppers?

They look like this. We have to do a lot of gas sampling and these are really difficult to assemble. I've been scouring the internet for advice but there are none. Do you have experience with ...
7
votes
1answer
1k views

How to reduce/eliminate cell clumping in suspension CHO cells, without using an anti clumping agent?

what is the best way to eliminate clumping suspension cells used for transfection. Anti clumping agents interfere transfection and hence can't be used. Though maintaining the cell density low helps, ...
2
votes
1answer
60 views

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?
19
votes
1answer
26k views

What is the difference between HPLC and FPLC and why is FPLC preferable for protein purification?

I've used HPLC (high performance liquid chromatography) before (once, so I'm barely even qualified to know what it stands for) so I was surprised when my labmate told me she would be using an ...
2
votes
1answer
1k views

Why does the pET- expression vector contain a LacI gene additionally to the one in the genome?

The pET plasmid is used for protein expression with T7 promotor in expression strains, such as E.coli BL21(DE3) It contains a lacI gene which codes for the lac repressor protein, a protein of ...
4
votes
1answer
430 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 ...
1
vote
1answer
1k views

RNeasy Mini Kit low 260/230 ratio — can I purify this RNA for further use?

I used Qiagen's RNeasy Mini Kit to isolate RNA from 5*10^5 C28/I2 (immortalized human chondrocytes). However, my yield is low (~25 ng/ul), but my 260/280 ratio is great (~2.3), and my 260/230 ratio is ...
8
votes
3answers
523 views

Does the Petri dish lid orientation on workbench affect aseptic technique?

When examining an incubated plate, it is rather clear that holding the plate in one hand and the lid in another is best for aseptic technique, or simply resting the lid on the plate as shown below. ...
3
votes
1answer
64 views

Non-fatal / low-harm measurement of Drosophila traits

I am trying to generate some candidate traits to measure in a fitness assay of wildtype outbred (lab population) flies. The key trait I will measure is lifespan. I am looking for some additional ...
13
votes
2answers
906 views

Are there issues with filling PCR tubes to capacity?

I'm planning to scale up a PCR reaction, and I'm wondering if filling the PCR tubes to the maximum volume of 200 ul would be a problem. It would mean a lot less pipetting as I would only need 1/4 of ...
6
votes
2answers
485 views

introduction to Chip Seq

I hope this question is suitable for this site. I am concerned about the Chip experiment part so I think it should be okay. I am a Applied Math student starting to get into bioinformatics and so I've ...
7
votes
4answers
377 views

Is there a comprehensive life science techniques/methods database?

There are so many techniques/methodologies in the life sciences that we can use to interrogate interesting questions. The thing is, most of us are completely unaware of the available methods we can ...
3
votes
1answer
165 views

PCR master mix contents

I am running PCR reactions with different sets of degenerate primers and I want to know what should go into the master mix My usual master mix: DEPC water PCR buffer (-Mg) 25mM MgCl2 10mM ...
2
votes
1answer
75 views

Do you have experience with PacBio?

I prepare a experiment and I found $PacBio SMRT$ as the great way to sequence my PCR products. I find the cost: library preparation 655 dollars + sequencing 435 dollars. It seems very low. Do you have ...
3
votes
1answer
228 views

How to measure quality and quantity of DNA?

I would like to mesure DNA. I quantify the concentration with Qubit fluorometer, but I would like to know also quality of DNA. I try BioAnalyzer (Agilent),but without success. Bioanalyzer measure DNA ...
2
votes
0answers
2k views

What are the roles of guanidine-HCl and ethanol in binding of DNA to silica?

I'm trying to understand how exactly the binding to silica gel (in kits) step works and I cannot find any papers which provide an explanation of the physics or chemistry; especially on the way that ...
2
votes
1answer
193 views

Substitute 25mM dNTPs mix with 10mM dNTPs

I need to make a solution of multiple compounds, one of them is dNTPs. The recipe calls for 20 μl 25 mM dNTPs in a 1250 μL master mix. Unfortunately I do not have it available at that concentration, ...
3
votes
1answer
41 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 ?
11
votes
2answers
7k views

How do I clean phenol contaminated RNA without losing any of the sample?

I recently extracted RNA from developing plant leaves for the first time, as part of a very long and intensive experiment. The samples were extremely precious because of the amount of effort that went ...
3
votes
0answers
193 views

Gibson assembly using NeBuilder

I am supposed to construct a plasmid that contains features from two other plasmids. My strategy is to generate three fragments form the two other plasmids. I was encouraged to try Gibson assembly, ...
2
votes
1answer
118 views

Plasmid maintenance

I have obtained some plasmids used as integration vectors, this question may apply to all plasmids. I would like to have a somewhat continuos source for these plasmids, let's say that the origin of ...
9
votes
2answers
956 views

Gloves for Cell Culture?

I always wear gloves when I'm doing cell culture. Moreover, I always spray my gloves with ethanol to disinfect, so that I don't contaminate everything. However, I recently heard the argument that ...
2
votes
1answer
69 views

Pippin prep kits - expiration date

Do you use Pippin prep? We would like to buy it, but we need more info. What is the expiration date of kits for Pippin prep? Thanks!
5
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1answer
2k views

How to avoid air bubbles while pipetting?

I get air bubbles while pipetting small volumes. How can I avoid them ?
3
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1answer
357 views

Benefits of CLARITY?

What are the benefits of CLARITY over this technique that was published more than a year earlier? Of course the second technique needs a fancier microscope that is likely more expensive and requires ...