Nature Network - Recent topics from Nature Protocols Discussion Forum

Repeat sequences (0 replies)

S.Rekha Reddy — Mon, 28 Apr 2008 15:26:36 -0000

Hai,
I want to analyse a region rich in repeat sequences… Can anyone tell me how to analyse the repeat sequences in genome??

Biotin solubility (0 replies)

sanjay ghosh — Mon, 28 Apr 2008 13:56:59 -0000

Im planning to do S1 affinity chromatography where the RNP complexes are to be eluted by 5mM d-biotin. However, I find that the solubility of biotin in water is around 1mM max. Is there a way to dissolve biotin to 5mM or is it that higher amounts of it gets dissolved in the elution buffer but not in water. Please suggest.

Jurkat cell line (0 replies)

Fabiano Pinheiro da Silva — Thu, 24 Apr 2008 23:16:43 -0000

Hello everybody,

I’m starting to work with Jurkat cells, but I never used them.
Can I consider these cells really naive T cells?
If they are tumors, aren’t these cells activated and differentiated towards a Th phenotype?
Can I induce Th1 polarization by culturing these cells with macrophages plus LPS?

Thanks for the help,

Fabiano

how to calculate the amino groups. (0 replies)

taqi ahmed khan — Mon, 21 Apr 2008 15:59:36 -0000

i am working on the topic of glycation of proteins.i have done the TNBS method for determining the amino groups present.i have made a standard plot by taking glcine.but i am now confused how to calculate the amino groups of my modified proteins.

penetration into mitochondria (0 replies)

Ivana Gadjanski — Thu, 10 Apr 2008 11:44:02 -0000

Hello,
does anybody have some advice how to increase the penetration of my anti-GFP antibody to the GFP-expressing mitochondria in the axons in the mouse spinal cord? In frozen sections. We tried different ways, such as using methanol, or mixture of methanol-chloroform…does not help…Any idea? Thanx a lot!
P.S. The Ab stains the axons, which also express GFP. So the Ab works, but it can not get into the mitos. And, I always use Triton X-100, as well

Purification Of Myc tag protein from bacteria (0 replies)

Rahul Saxena — Mon, 07 Apr 2008 20:46:27 -0000

Can anyone send me the protocol for purification of Myc tag protein from bacterial expression system..Thanku

No ligation!!!!!!! (1 reply)

Cinzia Pezzella — Sat, 29 Mar 2008 10:38:43 -0000

Hello, I’m new in this forum discussion and today I wanted to post my problem..then I discovered It was the same problem you have: no ligation and I can’t understand why!
As you, I’ve tried different DH5a batches, gel purified vector and insert, and tried up to 1:5 ratio in ligase reaction, but I can’t get non colonies. I’m using T4 DNA ligase from Roche,aliquotating the buffer to preserve ATP. I do the ligase reaction on 100 ng of vector in a final volume of 10 ul. The I’ve tried to transform 1, 3, 6 ul of ligase reaction in 50ul DH5a, but I can’t get non colonies. I have a negative control (ligase reaction without insert), but I can’t get colonies too.
These are the details of my cloning:
I’ve cloned FUR gene in pGEMTeasy vector, then digested this vector with AgeI and ClaI (within the FUR gene) to subclone another insert (LEU2,digested with compatible ends NarI, XmaI) in the FUR gene.
Both vector and fragments have been gel purified after digestion (Leu2 fragment comes from a pUC19 vector containing Leu2).
- Do I have to inactivate Ligase before transforming?
Please answer me!!!!

No ligation! Can't understand why! (1 reply)

Anu Ram — Fri, 28 Mar 2008 21:21:43 -0000

Hello everybody,

I am currently trying to ligate a _{80 bp duplex synthetic oligo (that has the 2 required restiction site over hangs) into a double digested 5.2 Kb plasmid vector using NheI and SacI. So far I have tried:}

1. 3 different T4 ligases!
2. Postive control that I know works (I have used the same vector and different insert (35 bp duplex) that I had cloned before).
3. After restriction digestion of the plasmid, I have tried both gel purification and without gel purification (tried eliminating the ~60 bp unwanted sequence by Microcon spin down; which should eliminate anything less than 200 bp according to the product catalog)
4. Apart from the 3x molar conc of the insert, I have also tried 10x,20x an 100x insert to vector ratio after reading some posts online…
5. Tried diffrent DH5a batches, and new ampicillin plates.

Even after all that, I have not gotten a single colony with my insert! Also, The synthetic oligo sequence has passed the HPLC quality control of the company and so, I think it is probably not the insert sequence issue.

I have spent about 2 months trying to do all this! I am completely bewildered and not sure what my next step should be…

I would appreciate any thoughts/suggestions that you may have.

Thanks in advance.

Na/K ATPase Western Blotting (0 replies)

Shina Menon — Wed, 26 Mar 2008 14:13:42 -0000

Hi
I am working on Na/K ATPase detection in kidney tissue by western blotting. Using the antibody specific for alpha-1 subunit, I am getting 2 distinct bands- one around 100 kDa and the other around 50 kDa. The 100 kDa one is where alpha subunit is supposed to be, but I am not sure how to interpret the second band. I even tried using 2 different methods of sample preparation (boiled vs unboiled) but the bands are pretty consistent.
I would appreciate suggestions on this topic.

yeast isolation (0 replies)

mustafa vohra — Tue, 25 Mar 2008 13:48:54 -0000

hello friends my topic for project is production of alcohol and poly alcohol from agricultural n industrail waste using yeast.so my first job is to isolate yeast from various source.well i started isolating yeast from various fruits by using gye agar.but the problems is that various other fungi except the yeast dominate the growth in plate.so what should i do to get specifically growth of yeast.any other media u can suggest or any ther help.i take glucose conc 20% while usin gye.thank u

trouble with acrylamide gel (4 replies)

Nisha padmanabhan — Thu, 20 Mar 2008 09:24:13 -0000

Hello,

I m having trouble running a acrylamide gel. My marker doesnt resolve at all. I have remade my acrylamide and yet still doesnt work. Any suggestions?

Thanks….

ligation and transformation problems! Urgent! (3 replies)

chunmei zhuang — Sun, 16 Mar 2008 22:29:11 -0000

Hi,
I am trying to ligate my gene 1400bp to a 6.4kb vector. I double digest the insert and vector from two plasmid with bglII and xho1. After that I purify the insert and vector for the gel with bio-rad kit or promega kit. Then I do the ligation with the different ratio insert: vector=3:1 or 5:1. The T4 DNA ligase I use is from invitrogen. The protocol for the ligation is room temperature 4h or 16 degree C or 4 degree C overnight. The problem is that I can get nothing after transformation. I tried many times, but still get nothing. I am nearly crazy now.
I did the positive control with PUC19. The efficiency of my competent cell from invitrogen (Subcloning Efficiency™ DH5α™ Competent Cells) is no problem. The transformation efficiency is 2×10E7 at least.
I did another test with the PBSK plasmid. Cut a single site with BamH1, and then I did three transformations with:
1. 1ul Digest solution
2. 1ul Digest solution(after inactive enzyme at 70 10min) add ligase at room temperature 3h.
3. After purification, 1ul product (DNA gel check the lane very well) add the ligase the same with 2).
The result was I can get nothing from 1) and 3), for the 2) I can get many many clones. The problem seems like is the purification problem, but I don’t know what the exactly problem is. I have used three different purification kits from different company. The 50xTAE stock solution and agarose I used is from fisher company.
Could anybody give me some suggestions? I would be very much appreciated if you could answer my questions!

protein expression in bacteria (3 replies)

Joao Goncalves — Fri, 14 Mar 2008 18:17:06 -0000

Hello,

I need to produce a protein in bacteria and have no experience in that field. I need the protein to produce an antibody against it.

I’ve cloned its coding sequence in the mammalian vector pcDNA3. This vector has the T7 promotor…does any one know if this is enough to express the protein in bacteria…or should I clone it in a proper bacterial vector?

Thanks in advance.

João

RT-PCR for a one-exon gene? (7 replies)

Eva Amsen — Mon, 10 Mar 2008 21:23:08 -0000

I’m trying to see if I get knockdown for a gene for which there is NO available antibody to the protein, and for which I can’t design any RT-PCR primers because there is only one exon (so I can’t differentiate between genomic DNA or cDNA)
(Actually, even ignoring the RT-rules, there doesn’t seem to be any suitable primer pair within this exon either.)

Is there anything I can do (within a few weeks) to monitor knockdown, preferably at the mRNA level? (I am certain that the knockdown construct is present, as it’s fluorescently labeled and I’m FACS sorting, so I “just” need to know that it’s actually working) Obviously, raising an antibody to the protein would work, but sadly, I don’t have that kind of time, and it’s not that important, and I don’t get enough lysate to look at protein levels anyway.

PCR optimaization (2 replies)

karthick Natarajan — Fri, 07 Mar 2008 14:04:07 -0000

hai..

any body give me suggestion to optimize PCR
i am getting bands of below 100bp only please procide some suggeation.
regards
karthick.N

TLR4 IP (0 replies)

Fabiano Pinheiro da Silva — Wed, 05 Mar 2008 01:02:32 -0000

Does anybody know if it’s possible to IP TLR4 in primary cells (macrophages).
People say the expression is low, so it’s very difficult to do that.
Does anybody know how I could do that? tags?

Thanks,

Fabiano

low molecular weight peptides and spin column purification (2 replies)

James Ghadiali — Mon, 03 Mar 2008 19:10:42 -0000

Has anyone had any luck desalting low molecular weight peptides (~2000Da) using spin columns with LMWCO gel filtration media like Sephadex G-10?
HPLC purification is posing some problems and we would rather avoid dialysis…

COMET ASSAY (1 reply)

farheen zehra — Mon, 03 Mar 2008 12:04:22 -0000

HI,
I am a young researcher and going to perform the comet assay for detecting DNA Damage in mussels {green shells)..
Can anyone help me how much time is appropriate for the electrophoresis that is for how long the current should be applied and appropriate voltage and should we have to put our electrophoresis unit under yellow light to avoid UV Damage of DNA..As we have to carry out all work under UV light.
I will be really thankful if anybody solve my problems regarding comet assay.
thanks

primary cilium (0 replies)

Joao Goncalves — Mon, 03 Mar 2008 09:49:11 -0000

Hello everyone,

Does anyone know if VERO cells can differentiate primary cilia? I can’t find anything on the internet so they probably don’t.

Thanks a lot.

Indo-1 calcium imaging (1 reply)

Gayatri Venkiteswaran — Sat, 01 Mar 2008 08:54:52 -0000

Hi, I am a Phd. student and my work is mostly calcium imaging in primary neuronal cultures. Lately, I have been trying to standardise imaging with Indo-1 AM.FYI, Indo-1 is a ratiometric single excitation, dual emission dye. So, exciting at 338 nm should give emission at 405nm ( which is the fluorescence of the Ca2+ bound form) and 485 nm (Ca2+ free form). Increasing cytosolic calcium would therefore cause an increase in the 405nm emission with a concomitant drop in the 485nm emission. However, increasing the calcium inside with ionomycin, i not only see an increase in the emission at 405 nm but also a small increase in 485 nm.i have checked the optics on my microscope and everything appears fine. Does any body have any suggestions?