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

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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, ...
4
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0answers
38 views

Can microdialysis be made in Drosophila melanogaster?

I've asked this before in stack overflow's cognitive science community, and someone recommended me to ask here: I've found a couple of studies using microdialysis on insects, but didn't found any in ...
3
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2answers
53 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?
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1answer
67 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, ...
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1answer
44 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 ...
2
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1answer
49 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 ...
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1answer
96 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 ...
13
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2answers
373 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 ...
2
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1answer
245 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 ...
5
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2answers
136 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 ...
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0answers
41 views

How to make a concentrated solution of the common lab glassware cleaner Alconox

I'm using the all famous 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 more friendly concentrated liquid to ...
3
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1answer
39 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 ...
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0answers
13 views

Derivative form of HRM results

I used in the laboratory the Rotor Gene 6000 for a real time PCR and HRM analysis. When i analyzed the results of melting step, the derivative plot in the y-axis used the dF/dT . On the other hand ...
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1answer
56 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 ...
2
votes
1answer
69 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
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0answers
193 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
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1answer
65 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
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1answer
24 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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2answers
820 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 ...
2
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0answers
86 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
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1answer
76 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 ...
7
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2answers
378 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 ...
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1answer
42 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
457 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
101 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 ...
3
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1answer
28 views

Prevalent large (>=90kDa) maintenance protein/loading control

I was wondering if anyone had recommendations for good, large (hopefully 100kDa+), control proteins that would be present in most mammalian cells. I'm working mostly with tissue samples from humans ...
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1answer
54 views

In vitro transcription, contamination problem

I am using a RNA which is in vitro transcripted before I started my project. It turned out it is not prepared properly and has DNA contamination. Instead of perform the in vitro transcription again, ...
3
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1answer
156 views

For people who work in microbiology labs, what software do you use to manage your strains?

It's just a small microbiology lab that currently records everything on paper, and there's quite few mutants as well. Is Excel commonly used for this sort of thing? Or is there a better software to ...
4
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4answers
124 views

Electronic laboratory notebook (ELN)

I have been using old style lab book for some time now but with increasing work on computer and storing sequencing results and gel pictures on computer it would be nice to have everything on computer ...
2
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3answers
109 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
424 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 ...
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0answers
12 views

Gas sampling, in need of large septums

I'm designing an experiment that will require many gas samples taken from sealed pipes with a 1" (2.54 cm) diameter. I have been searching for large septums but have had no luck in finding a size ...
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0answers
24 views

Improving throughput of CFU plating?

In a separate question I've described my general experimental setup where I need to measure the number of live cells in a growing bacterial culture in a fairly rapid and high-throughput manner. In ...
6
votes
1answer
250 views

Alternatives to CFU plating for measuring number of viable cells?

I am hoping to measure growth rates of a bacterial culture in several growth conditions. I am concerned that these growth conditions may cause cell death, which would lead to a decreased ...
4
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1answer
743 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 ...
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0answers
88 views

Which is the best way to fix and preserve comet assay slides before staining with Ethidium Bromide?

I want to use the method of Single Cell Gel Electrophoresis (comet assay). I have many samples, but I don't have the appropriate microscope. So I have to preserve my slides until the time comes for ...
5
votes
2answers
100 views

Measuring fitness / lifetime reproductive success (LRS) in Drosophila

I am planning a fitness assay of Drosophila melanogaster. I'd like to get a good measure of lifetime reproductive success (lrs) but I don't want to count all the offspring produced over a lifetime by ...
3
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1answer
55 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 ...
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1answer
45 views

Enzymatic Protein Deglycosylation of TCL

I've done O- and N- linked enzymatic deglycosylation of purified and lyophilized proteins. Following some modifications to the manufacture's protocol, I had great results. I now want a student to ...
2
votes
1answer
95 views

Should the length of the electrodes in the electrophoresis chamber be proportional to chamber's size?

I am trying to build a small horizontal electrophoresis chamber from scratch. I want to use it for comet assay and I will be using only 1 slide, so it's going to be about 3cm wide, 10cm long and 4cm ...
2
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0answers
42 views

Removal of the Initial Methionine in Venus for FRET

I'm working on building some FRET reporters. In addition to a cleavage site (of varying composition from 15-18AA), a 1 AA linker, I'm using Venus and Cerulean. Initially I was worried that 18AA ...
3
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1answer
58 views

What makes drosophila eyes red? and is it stable?

I have Drosophila melanogaster which I am doing an eye pigmentation assay on in the future. To do this I will dissolve the heads, 10 of them removed from frozen whole flies, in acidified ethanol for ...
8
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3answers
206 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. ...
4
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2answers
565 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 ...
7
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1answer
213 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
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0answers
123 views

What tests can be performed to test the purity and quality of the raw peptide HCG (Human Chorionic Gonadotropin)

What tests could be run to test the purity and type of HCG? We are looking to purchase HCG from China but the purity and quality varies between labs, we are able to receive samples of the raw peptide ...
5
votes
1answer
55 views

What method would you use to genotype SNPs in low quality samples?

What method would you use to genotype SNPs in low quality samples? I ideally want to genotype hundreds of SNPs in hundreds of scat samples (very low amount of target DNA, potentially degraded and ...
4
votes
2answers
311 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 ...
3
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1answer
104 views

How do different tissue culture matrices affect background in fluorescent microscopy?

In response to my previous question, I've been reading up a little bit on poly-D-lysine, Collagen I, Collagen IV, laminin, and other tissue culture coatings that promote cell adhesion. I've always ...
2
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1answer
463 views

Optogenetics - How do microbial opsins work?

I'm just introduced to the optogenetics method and am having some trouble grasping the genetics (of the optogenteics) part of things. So we have Retinal and Opsin that form Rhodopsin molecule that ...