science - biology | RSS MAD BioForum Method Discussion

problem using IPL 41 medium

Hello everyone,

I am using IPL 41 medium with 10% FBS for culturing Sf9 cells. The problem is a lot of crystal - like deposits in the plate within 48 h. Why is this happening? I don't add yeastolate - can this contribute to the deposition I have observed?

Thanks in advance for all your suggestions...

ProteoJuice Protein Transfection

Dear all, I am using ProteoJuice protein transfection kit to transfect my protein of interest into MCF7 mammalian cells. I have tried many times with but seems like the protein couldn't be transfected into the cells. Has anyone use this kit before? Any suggestion for me? Or is there any good protein transfection kit? Thank you.

Zebrafish in situ hybridization

Hi all,

I have a few questions regarding to zebrafish whole mount in situ hybridization, hope someone can help me. I'm confusing with too many different protocols available online.

1) Deionized formamide or formamide in hybridization buffer? Why should we use deionized formamide but not formamide?

2) I purchased yeast tRNA from Sigma in powder form. Should i extract RNA from it or use it directly in hybridization buffer?

3) How to store DIG-labeled RNA probes (600 bp) correctly so that it will last long? Is RNase needed in probe storing? Should i add the probe in hybridization buffer once it is synthesized to store it?

4) If the sense sample has a same staining pattern as the antisense sample, what does this mean? (I'm sure i didn't mix up both anti and sense probes and I got same result after repeating twice. I got my staining after overnight which is weird as normally i get my staining within 1-2 hours. Nothing change here. Could it be probe degradation?).

5) I need to perform ISH in various stages of embryos. Other than different proteinase K treatment time, any other parameter i should change to optimize for each stages?

Thanks in advance. Sorry for taking your time but I really have lots of questions for ISH.

Kuahmk

Cell clumping in culture: Folic acid or i-Inositol?

Hi,
I am using a medium that I prepared by myself according to the description sheet of the producing company (being cheaper that way), but I am getting abnormal cell clumping on the very next day. The original medium is Myelocult, and it contains MEM, Folic Acid, i-Inositol, L-glutamine, FBS, Horse serum, 2-Mercaptoethanol. When I cultured same cells from same sample with RPMI medium, and the prepared medium, clumping occured in the prepared medium only. I am guessing it is either the Folic acid or the i-Inositol, being the 2 constituents supplied in powder, and the weighing was difficult.
Do you know which of these 2 would cause cell clumping if increased or decreased in the culture medium??
Thanks

experimental determination of protein function

Hi there:

I would like to know what type of experimnts can i use for determining the function of my protein?
My protein is complemnt facor B (CFB) involved in the alternative pathway of complement system to destroy pathogen.

Is the two yeast hybrid system can be used for determination of CFB function. Am i right in my thought.
Is there any alternative experiment to determine CFB function?

Thanx

looking for a 2-in-1 kit for Genomic DNA/RNA

Hi everybody, I am looking for a 2-in-1 kit for Genomic DNA/RNA. Can you give me some idea? Thanks a lot!

looking for Genomic DNA/RNA kit

Hi everybody, I am looking for a 2-in-1 kit for Genomic DNA/RNA. Can you give me some idea? Thanks a lot!

qRT-PCR first timer, controls?

Hi everyone,

I am new to RT PCR. I am using RNAeasy to make RNA, qscript to make cDNA and I made primers using this freeware http://www.idtdna.com/Scitools/Application...nceWarning=True (specific for RT-PCR). I BLAST the product I would get and it was highly specific for just my gene of interest in my species of interest.

I didn't run the RT, a friend did using SYBR green. It worked quite nicely and I got nice reproducible (made 3 batches of cDNA from each cell line) results. HOWEVER, I didn't run any controls, and I don't know what controls to run. (normalized everything to GAPDH)

After the run I ran some of the PCR reaction on a gel and only got one product. I think this is a good sign.

The only control I've found here is to maybe run a PCR on the mRNA I made to make sure it isn't contaminated with gDNA. When I quantified the RNA after making it my ratios were quite nice (all between 1.8-2.2) so I'm assuming it is clean.

Is there anything else I can do, any big controls I'm missing?

how to see A beta by western blot

Hi all
I am trying to see abta 40 and 42 by western blot using tricine gels, but still the bands are not clear and most of the time the protein aggregates. Do you know any reliable method or any trick to avoid aggregation and see the monomers clearly?

Thanks soooo much

Combinatorial RNAi - double gene knockdown

Hi ,

Please tell me what to do -

I am trying to knockdown 2 proteins together - so i want to use 2 different RNAi's together at the same time - i have decided to split them in their concentrations and bring them upto 100 nm and am using the dharmafect reagent for transfection...and would be transfecting them twice . Can i increase their concentrations like use 100 nm of each at the same time is that possible...?????????
Has anyone tried something like this before..please let me know your comments or anything else i can do ???

It is nessesary to apply aseptic technique for PCR?

I think that the reason I had multiple bands in my PCR products is because of nuclease contamination. The enzyme entered the mixture and cut the DNA in various places, resulting in multiple fragments instead of the just the one bordered by the primers I used. I figured that the nuclease must have come from the contaminating microbes or skin cells from my fingers as I rub my fingers opening and closing the tubes. So whenever I mix the reagents, I do it under the laminar flow, while wearing gloves sprayed with disinfectant.

Is it really nessesary?Or should I just assume instead it's due to the wrong annealing temperature (yeah I got the multiple bands again).

Promoter analysis of Cyclin D1 and across species

Hi

I am interested in looking for the promoter of Cyclin D1 and compare that across species. I would be very grateful if anyone here could provide me with any website where not only can I find the promoter of the gene of my interest as well as would be able to compare it across the species.

thanking you all in advance!

FCS without LDL

Hi everybody!

can anybody tell me if any company has FCS without LDL?

Thankyou for your time.

Liagtion based technologies for SNP genotyping

Hi All,

I am using oligonucleotide ligation assay (OLA) for SNP genotyping. I came across other ligation based chemistries such as PCR\LDR assay that can be used for SNP genotyping.

Are these two technologies different? They appear to work on the same principle. PCR\LDR uses a set of two SNP specific primers (located at their 3' end, wild and mutant) and a common primer. Similarly, OLA also uses two SNP specific probes and a common reporter probe. Could someone point me to the design parameters and considerations used for each?

Liagtion based technologies for SNP genotyping

Hi All,

I am using oligonucleotide ligation assay (OLA) for SNP genotyping. I came across other ligation based chemistries such as PCR\LDR assay that can be used for SNP genotyping.

Are these two technologies different? They appear to work on the same principle. PCR\LDR uses a set of two SNP specific primers (located at their 3' end, wild and mutant) and a common primer. Similarly, OLA also uses two SNP specific probes and a common reporter probe. Could someone point me to the design parameters and considerations used for each?

Us of LiCl

Dear all,

could somebody please explain to me the difference in the use of lithium chloride instead of sodium chloride in e.g. IP wash buffers....

thanks a lot :-)

kylvalda

Na Acetate buffer recipe

Hello Everyone,

I am a bit confused in preparing acetate buffer using na acetate and acetic acid. i need to prepare 10 mM na acetate buffer pH 4.0 and 25 mM Na Acetate buffer ph 4.5 and one with 25 mM Na Acetate buffer ph 4.5 containing 0.4 M Nacl. how much quantity of sodium acetate and acetic acid do i have to add to get the exact pH. can i prepare concentrated solutions and later dilute them as per the use.

kindly guide me on this

thanks in anticipation

exec

Coupling Agent

Does anyone know ANYTHING about the coupling agent "Carbonyldiimidazole" ... i.e. how it works and the conditions (pH, buffers, etc) that it works at? It seems to work by acting on a carboxyl group and an amine group liking the two via an amide bond... but if anyone knows anything further about the mechanism of activation it would be EXTREMELY appreciated.

Thanks in advance...

how to check organ(tissue)-specific expression profile of a gene

hello,
a basic question, I am doing westernblot of myocardin and I need to choose the positive and negative control.
so how to know its expression level at different organs or tissues so as to choose negative and positive control.
Thanks a lot!

isolation of phage lysogens

I'm trying to isolate lysogens (salmonella) of phages.
Whenever I try, I found lysogen is mixed with non-lysogenic strains when checked by PCR.

I steaked from the center of a plaque
--> streak to isolate single colonies : checked with PCR (1 positive from 3 single colonies)
--> restreak the positive colony : checked with PCR (2 positives from 12 single colonies)
---> restreak the positive colony : checked with PCR (1 positive from 12 single colonies)

Why are still there so many non-lysogen strains on the plate? Do I need further streaking to isolate lysogenic strain?

Please help. Thank you in advance.

2-D gel analysis

Hi..

Does anybody know how to find the whole saliva 2-D reference gel?

TQ...

Enzyme Unit

Hi, If I'm using mushroom tyrosinase (25KU), lyophilized powder form (Sigma), the enzyme activity is ≥1000 units/mg solid, how to prepare different concentration of mushroom tyrosinase in unit U/ml? I want to prepare into 25, 50, 75, 100, 200, 300, 400, 500 and 600 U/ml.

Thanks.

PCR

Hi...
I run a PCR test for an infectious disease. When the negative control became positive the steps to follows (according to my boss)are:

1st. Re run the negative control (the contaminated one) in my next run.
2nd. If the control is Negative on the repeat, go back and accept all samples from the original run.

Any one would like to discuss this?

I think that the run is to be invalidated and then all samples to be repeated using the same original sample and starting with DNA isolation all the way to amplification .

I'd like to know any other people opinion about this.
Thank you so much for your time.

Floppy

Any better reagent for isolation of PBMC from whole blood?

Hi. I am trying to find a better reagent for the isolation of PBMC from whole blood. I am currently using Ficoll-paque PREMIUM, which is very frustrating as the PBMC gets contaminated with red blood cells. Can anyone help me with this? Thank you.

siRNA transfection: DNA conc optimization problem

Hello All
im using Cho-K cell line for transfection studies. previously i did optimized DNA conc. which was 50ng (for RT-PCR) using Lipofectamine 2000. but now when im using siRNA for knockdown analysis. im getting nothing, would anybody tell me where im wrong or what could be the possible reasons for not getting RT expression????????????????
looking forward
thanks
Bushra

Sodium azide

How much sodium azide should you add to PBS to preserve blots that you have already probed?

LOWRY ASSAY

Hi all,

Everytime I conduct Lowry Assay, the BSA not dissolved properly in the 0.5 M NaOH. The BSA (Sigma) become a gel-like clump at the bottom of the test tube. Is it normal or there are something wrong with my BSA? Is Lowry Assay is the best method to estimate protein concentration or should I change to other method such as Bradford Assay?

Thank You

Homebrew Gel Documentation System - Burnout of Digital Camera?

I threw together a 'gel documentation system' using an old difital camera (I was sick of the stinky polaroid). I made an adapter out of card board so I could place the digital camera where the old polaroid sat. Works pretty good, gives a fire color through the yellow filter!

Anyway, it seems to be fading?
It fades markedly while taking pictures in one sitting. Below is a picture (15second shutter) that took immediately, then the other picture is one I took with the same settings after taking a few with different shutter times.

Does anyone know anything about digital cameras, UV, ...? What's going on here?

Getting RNA and Protein out of/off from Matrigel

Hi everyone,

I am trying to figure out a way to get cells out of Matrigel after they have undergone channel formation to try and run PCR and Western blots. Does anyone know how to get the cells off without taking a bunch of Matigel off with them, or some way to make a cell pellet from this? I just keep getting Matrigel in with the cells when I look at them under the microscope. Thanks very much.

Lauren

Transient transfection with a pcDNA plasmid

Hello, I am doing transient transfections with a pcDNA3 plasmid, after having trouble with my stable cell lines. The response I see with this plasmid in a transient transfection is great, but now, I would like to know if I'm looking at chromosomal integration and not just protein expressed transiently like with a transient vector.

I transfect my cells with lipofectamine for 4 hours, I serum starve for 24, dose with compounds and on the third day I measure gene response. Was that enough time for chromosomal integration?
Is the pcDNA3 vector has the ability of transcribe a protein without integration? like a normal vector would?

Can anyone can help me?
sorry for all the questions
Thanks

help please, RNA storage

Hello,

I am currently working on RNA extraction from bacteria, I need to transfer my extracted RNA to another lab what do you think I should use, should I freeze them with nitrogen, purchase special containers to storage and transfer them? any recommendations?

thanks a lot

How to detect the presence of Poly(I:C)?

I am using Poly(I:C) as virus mimicry in the IFN experiments. I wonder if there is any method by which I can show Poly(I:C) does enter the animal. For genuine virus, I think I can use PCR to amplify a species specific sequence. But what can I do for Poly(I:C)?
Any suggestion is welcome. Thanks.

apoptosis and PS exposure

May I ask whether all apoptosis will show annexin V positive ? I did some annexin V staining but I couldn't see any PS translocation. Does anyone know which caspase or enzyme is responsible for PS translocation? Thanks!

Nuclear protein extraction of worm

Hi,

I woud like to extract a nuclear protein from whole C.elegans worm.
I know how to obtain a nucleus' pellet ... but after?
does anyone have a protocol for this?
Thanks,

Zekio

Nuclear protein extraction of worm

Hi,

I woud like to extract a nuclear protein from whole C.elegans worm.
I know how to obtain a nucleus' pellet ... but after?
does anyone have a protocol for this?
Thanks,

Zekio

Which Kit should I use for microRNA microarray?

Hi All
I am a new one involve in MicroRNA.
I want to do a embryo tissue microRNA microarray.
Any suggestion about which Kit I should use for microRNA microarray?

Some body mentioned Abicom. Recently, invitrogen and sigma claimed that their kits are better. Any suggention about that?

Thanks in advance.

zeno

CDK6 kinase assay

Dear all

I would need your help and all suggestions for doing a kinase assay in-vitro to detect the activity of CDK6. I have never done a kinase assay and as I see the papers published, they have all used only radioactive detection method!!. It would be of great help if anyone around has had experience in kinase assay preferably in looking for the CDK6 or similar kinase activities.

all help and suggestions are highly appreciated and I would be deeply grateful for them

a protocol and also if possible a source from where to get the reagents can help a lot more..

thanking you all for your time and help

best of things

Can the current technology make a human cell solely using materials from nature?

Striatal and/or SN neuronal primary culture

Hello everyone!
do somenone knows a protocol on how to prepare primary dissociated neuronal cultures of striatum or SN? I currently have experience on cortical and hippocampal cultures from P0-P2 rats, so it would be better to me to use postnatal brains instead of embryonic. I've found a lot of protocol on E15-E19 rats but almost no-one on P0-P2... why is that? Dopaminergic neuron doesn't grow from this stage?
Thanks a lot for all the help and suggestion

Template-switching with reverse transcriptases

Hi all,

I am attempting to do "template switching" in my reverse transcriptase reaction, using a template-switch primer with "GGG" at the 3' end. I was hoping to incorporate this primer sequence at the 5' end of the cDNA (that is, 5' relative to the orientation of the mRNA).

I am using Superscript III at 50 or 55 degrees C but so far couldn't detect a template-switched product. I even included a 5-minute room-temperature incubation in the middle of the RT reaction to allow the GGG to aneal to the CCC overhang left by the RT. I also tried adding 2mM manganese at the end of the incubation to decrease the template-specificity of the RT (I don't know whether this really works, but there was one paper I found where they did that). There is plenty of cDNA in the end, but I don't think there is any with my primer sequence at the 5' end, when I assayed by PCR.

I have not heard about anyone else doing template switching using superscript III, is it possible? Does SSIII leave a C(3) overhang when it comes to the end of the template RNA? Or does the high temperature destabilize the base-pairing between primer and cDNA?

Any help would be appreciated.

count cell number

Hy boys!
How can i count the number of EUKARYOTIC cells in colture, using a spectrophotometer setted to 230, 260, 280, 320, 595 and 600 nm?
Please help me...
Thanks

384 well plate

please share your experience/disadvantages to use 384 well plate for real time PCR

Could we grwo chinese hamster cells in untreated normal petri dishes?

I am doinig an antibiotic selection with g418 on my adherent chinese hamster cells. i need my cells to grow slowly and for a long time. can i use normal untreated petri dish instead the cell culture treated plates in which the cells grow like mad? Also could the material at the bottom of the treated ones detoxify the antibiotic? I am using v79 derived cells that are adherant type.

Thanks
Aby

Strange large bands on my PCR gel, has anyone seen this before?

Hi,

Im getting strange genomic sized bands (gel 1), any ideas on what is causing this and is it related to the failed reactions? I am trying to amplify the ras-related protein gene of Phytopthora(~400bp). I have tried ethanol precipitation and diluting the DNA (1:1, 1:5 &1:10) and the large bands are still there!! Any help would be greatly appreciated as it is difficult to get more cultures for these samples plus it is driving me to the bad karma hypothesis!!! I have also attached the DNA gel (gel 2).

Cheers,

Matt

Could the strange G418 resistance fo my Chinese hamser cell lines be due to the

I am having a hard time with G418 resistance of my Chinese hamstr cell lines in a HOMOLOGOUS RECOMBINATION ASSAY. The cells are resistant even to a very high concentrations of g418. The conc. for selection is 1mg/ml in most of the papers that are performing this assay. Could the tissue culture treated dishes contain some detoxyfying protein that inactivates our G418 antibiotic? In the papers they have used 100 mm petri dishes. But they haven't said that they are tissue culture treaed or untreaed. Should i use the untreated ones for antibiotic selection?

control sample as a reference for Revers-phase HPLC

Dear Friends,

I want to use Reverse-Phase HPLC to separate my bacteriocin which it was extracted from free cell supernatant of overnight culture of my lactobacillus acidophilus.

I want to know what would be my control sample since I am working with unknown bacteriocin and I don't know what type bacteriocin or in what molecular weight range is inside of the extracted sample? is there any Standards available as control for this prupose in the Market for example for SDS-PAGE we have Protein standards with different range of molecular weight but how about if by RP-HPLC we want to find unknown bacteriocin? [I suspect that my bacteriocin is in low molecular weight (below 10 KD)]. what is control to run experiment to define it? do you have any idea about it since I'm new in this area. Thank you very much.

Leo

Etbr Disposal

Hi..
Might be funny.. what can be the safest way to dispose Etbr..To dispose gel and the gel running buffer

Primer design help - secondary structures

Hello there! Anyone care to help a noob out?

Basically theres an element within of a protein that we are interested in and in order to study that, Ive been asked to design primers (include restriction sites) to generate a variety of DNA fragments to use as inserts into vectors. Think of it as somewhat of an elimination process where certain regions of the cDNA are excluded and the consequences observed when that particular fragment is digested and ligated into a vector.

My understanding of the primer designing process with regards to this is that it is a fairly strict process. For example if I am interested in creating a fragment that will eventually be translated into residues 20-300 of my protein, I will need a forward primer that looks like this:

5 - NNNN Restriction site region of overlap of approx 15 bp starting with the first nucleotide of residue #20 - 3

So I came up with the sequences to my primers, and I put them through the Net Primer to check for any secondary structures etc To my horror they all came up with multiple putative hairpin structures, self dimerization, cross dimerization and at that point my hair was starting to turn grey.

Now its not like I input my DNA sequence into a primer designing program and let them spit out the best possible primers matches, since I need them to generate specific regions of the protein I dont have the flexibility to choose another part of the DNA that will give me primers with less secondary structures/lower Tm.

Are there any solutions to this? Or should I just go ahead and deal with it? OR is my understanding of this primer designing business is completely WHACK? (i.e. if Im interested in aa residues 20-300, I dont necessarily need to have my 15bp overlap starting exactly on the first nucleotide of residue #20)

And if you have read this far, thanks! Heh.

Need your suggestion about hybridization solution

I would like to know the principle of hybridization solution. Is it different between radiactive and chemiluminescence detection system? If I use chemiluminescence as a detection system, can I apply the the reagent from radiactive detection in my system?
Anyone help me please. Thank you for your kind suggestions.

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