BioForum

Sequencing with ABI Prism 310

Mon, 06 Jul 2009 05:04:09 -0700

Hi

We are using in our lab ABI Prism 310 Genetic Analyzer and I want to ask about capilares. ABI offer two types of capilares: 47cm for POP-4 polymer and 61cm for POP-6 polymer. For me most important thing is that I want to read first 300 nt of DNAs. Which capilares have better read resolution for the first 300 nucleotides?

Anyone can help me with this?

Best regards

Leszek

i'm so fed up and confused!!

Mon, 06 Jul 2009 02:57:04 -0700

Hi there everyone,
I think I need a bit of advice! I'm in my final year of PhD land, with 3 months of money left, i'm still running experiments in the lab and am really struggling to try and write up something at the same time! (and still have no papers out on my actual research as nothing is quite completed - I feel like i am really bad at the bench!!!)

The lab I work in is where I carried out my honours project, and I really like my supervisor and his wife (not as my boss's so much but socially they are fine!). The lab used to be pretty big, but when I came back afew years later after some time out there was really only me! So throughout my PhD i've had no post-docs or tech support in my area and have just muddled through with all the experiments kind of self teaching! Also, because its just me, and i have a seriously problem saying NO to stuff i've ended up doing so much other things as part of other smaller studies and I feel like my own project has suffered quite a bit for that! (we had a grant for my project to take on and do loads more stuff with post-docs etc, and my bosses have basically managed to get most of it done just by me! i'm shattered!!!!). I feel like because there's so much quantity of little things, the quality of the main stuff has suffered and I dont feel confident about my work, the results or anything! Like I said I really feel like I am not good at bench science, and I don't trust results or anything I do anymore. I have lost all confidence and basically just want to stay home all day and stare at the wall!
Anyway, i'm blabbering abit now sorry! My point is, this whole experience has really put me off working in science for the rest of my life. I know other labs may be different with more people, more money etc, however when I look at senior academics who spend long hours/weekends doing work and never getting any decent breaks I really dont think i want that for my life! I have recently applied for a job as a part time lecturer at a college, and my experience throughout my phD stands me in good stead (i'll let you know if i get an interview). Really though I dont know what I want to do anymore, but I really feel I can't quit at this late stage, and so many people are looking up to me to get the work finished, write a good dissertation (and family to be Dr!!! ). My partner asked me seriously the other night if i wanted to quit and really I do, but its just too late, I should have done it in first year.
Also, If i get my PhD and then decide to leave academia then my chances of getting a job are slim as i'll be TOO experienced for stuff, is that really the case, as I really see my future as a less stressful job with better hours and plenty of time for family and a social life.
Apologies for the long post, I think I could go on forever I just have so much on my chest, and my partner is fed up of hearing about it!
Any advice or just support would be really appreciated,
Cheers, KT

In vitro expression extracts

Sun, 05 Jul 2009 23:56:12 -0700

Hello everyone,

I am going to order two different extracts for my work:

PURExpress� In Vitro Protein Synthesis Kit from New England Biolabs
cat# E6800S 10 reactions (25 ul vol) USD$220.00

S30 E.coli extract system for Linear Templates from Promega
cat# L1030 30 reactions 286.00pounds

Has anyone used the above before? What is the difference between the two? I have been using Promega, and I find it a hassle to add the ingredients one by one. Is there any difference in the efficiency of the above two systems? Thanks in advance

EDTA as denaturating agent ??

Sun, 05 Jul 2009 22:21:15 -0700

hi

how effective is EDTA as denaturing agent for proteins???

GST pull down assay

Sun, 05 Jul 2009 19:31:16 -0700

Hi everyone.
I am currently using the GST pull down technique to validate protein interactions between two of my proteins. I am expressing one protein in HEK293 and the other in ecoli cells. I am currently using frozen lysates. Will this affect my interaction studies?

strange dots in cell culture flask??

Sun, 05 Jul 2009 19:08:17 -0700

Hi,

This is probably a stupid question... I have recently purchases a U87 cell line and I'm noticing some white spots in my cultures when viewed with the naked eye - I have not previously seen this characteristic when I had cultures of the same cell line a while ago. I think it's probably normal, as when viewed under the micro, it seems as though it's the natural way these cells grow. I have attached a pic. They also appear to have a slightly lower adherence compared to before. Could this be due to trypsining for too long? If so, what can I do to restore them?

Thanks,

any help would be greatly appreciated.

window of Mapmaker is too small!

Sun, 05 Jul 2009 18:42:41 -0700

Dear all,
As a new user of Mapmaker 3.0, I feel frustrated by its small window in PC, that is to say, when I type more commands I will no longer see the commands (and the results output) I typed before because they soon moved above beyond the window. Can anyone help me to solve the problem? Great thanks!
PS: I previously use mac OS to run mapmaker,but it seems that the new Leopord for Mac no longer support Mapmkaer. Can anyone tell me if there is any good application like Mappmaker work under Leopord? Thanks!

Sterilize DNA preps

Sun, 05 Jul 2009 10:51:06 -0700

Hi everyone,
I`m trying to transfect some cells with my DNA prep but I realized that it is contaminated.
How do you sterilized your DNA preps to transfect? Can I filter it on a 0,22um?

Thx,
DVD

amount of proteins in urine

Sun, 05 Jul 2009 10:10:08 -0700

hi all

can anybody tell me the amount of proteins excreted in urine of normal beings? is lowry a good method for detecting urinary proteins?
help me please....

About post doc

Sun, 05 Jul 2009 09:14:15 -0700

Dear Colleagues
I have selected some groups for my post doc, but I am very reluctant to contact with them. I can write an email but I donot know what should be the format of the email, can some one give me advice, what should I write, do I need to wrtite a cover letter.
How long should be my CV. Does one page CV is enough.
I shall be very thankful to you if you can advice me some thing about it.
with best regards

Vibrio anguillarum

Sun, 05 Jul 2009 09:03:32 -0700

Hallo all,

I am doing some literature review on Vibrio anguillarum and the disease Vibrosis and I was wondering if anyone here has a must have book or article in mind that is really important to have.

There is a lot of info out there, but maybe someone here has an important must have title, book, article, website in mind.

thanks.

Luciferase reporter induced by betagal vector??

Sun, 05 Jul 2009 05:32:03 -0700

Hi!

I'm trying to optimize my reporter assay with 3 vectors, expressionvector with transcription factor, reporter vector with luciferase and betagal vector.

I found that the luciferase activity (counts per second) is much higher whan betagal is also transfected, compared to just the other two vectors. I'm confused..... does betagal activates my reporter, or what?

the ratio between vehicle treated transfected cells and ligand treated transfected cells is almost the same though, but the numbers like 5 times higher for both when betagal is expressed.

using 0,5 �g of reporter and betagal, and 0,1 �g of expression vector with my receptor, for 500 000 HepG2 cells.

Greatful for any advice!! :-)

Microbial Carbon and Nitrogen

Sun, 05 Jul 2009 04:51:35 -0700

I'm trying to measure microbial C and N from soil samples using the chloroform technique. I've tried both the CHCl3-fumigation and direct addition with CHCl3 technique but unable to detect any differences between the control (unfumigated samples) and fumigated samples. I've tried incubating the samples for 1d, 5d, and 14d but unable to detect any differences. I'm analysing the samples using the titration method for microbial C and spectrophotometer for microbial N as i don't have acces to a TOC analyser. Running out of ideas here.
Most protocols mentioned that EtOH-free CHCl3 has to be used. How important is it and why? Please advice. Also, what is the simplest method for removing EtOH from CHCl3? Clearly i must be doing something wrong. would really appreciate it if anyone can provide me with a working methodology that yields results....desperately need help as time is of the essence here.

It's TJ's B-day

Sat, 04 Jul 2009 21:16:03 -0700

and for our:

Happy Birthday, Hombre.....

btw,the bash will come later and that's a promise

silver staining (dendritic spine visualization) protocol?

Sat, 04 Jul 2009 21:06:13 -0700

Does anyone have a silver staining protocol for visualizing dendritic spines with light microscopy? This is one I found at (http://synapses.clm.utexas.edu/learn/visualize/visualize.stm), any alterations or important notes anyone could suggest? I mainly do extracellular in vivo cortical recording and stimulating, not much experience with staining.

The classical Golgi impregnation method is very elegant in its simplicity:
1. Immerse a block (approx. 10x5 mm) of formol-fixed (or paraformaldehyde- glutaraldehyde-pefused) brain tissue into a 2% aqueous solution of potassium dichromate for 2 days
2. Dry the block shortly with filter paper.
3. Immerse the block into a 2% aqueous solution of silver nitrate for another 2 days.
4. Cut sections approx. 20-100 �m thick.
5. Dehydrate quickly in ethanol, clear and mount (e.g., into Depex).

thanks!

cell floating in 96-well plate

Sat, 04 Jul 2009 20:26:19 -0700

Hi all, recently I have encountered a problem on culturing RAW264.7 in 96-well plate. I need to do a fluorometric experiment.

here is my protocol:

Day 1: seed 2x105 cell into 96-well plate. Maintain it O/N in DMEM (no serum).

Day 2: remove medium (i tried using a pump or pepitte), add back fresh DMEM (no serum).

The problem is at step 2, the cells start to detach once I added the medium. I cannot do any washing in the downstream steps.

I don't think there is a problem with the coating. I routinely use the one from Corning and try plates from other labs which they have no probelm.

What else could cause the problem? (e.g. passage number? the cell is at 24th passage)

Thx

A Diploma in biotherapeutics from reliance life sciences?

Sat, 04 Jul 2009 12:54:12 -0700

Hi.......... I am a Bachelor of engineering in Biotechnology (BE biotechnology) from India. I have just now completed my studies and have been planning to do my masters, but its too late to apply here in india for this year so i will have to try again next year.

In the meantime i have been selected for a diploma course at reliance life sciences(mumbai), i was selected throught a written test and interview. The course duration is onw year and only 20 students are admitted in a year. Further info of what the course is in this link http://www.rils.ac.in/html/adp.html , i have been selected for the " Diploma in Biotherapeutics" program...

The course fee is 1lakh rupees, my problem is i wanted to know if i can get a job abroad with the help of this course? are there companies who will be willing to take me if i complete this course? As the main focus of the program will be in stem cells, will i have other future options??

Please help me,

Thanking you
Ponybn

cDNA

Sat, 04 Jul 2009 08:34:14 -0700

Hi ev1,

Can anyone help me out? I'm having problem analysing my cDNA.

I used to run PCR straight after RT but i got no bands for result. so, my friend suggested i run a gel on the cDNA prod.i got nice band but i'm not sure whether it is the my cDNA prod. To normalise it, I tried running housekeeping genes of GAPDH, GST, cytp450 as positive control but all show no band. Thus, i cant prove tht the band was my expected cDNA. What should i do? I cant figure out what has gone wrong..

Has anyone try running cDNA on the gel? What ladder marker u guys used? Is it the cDNA expected size shld b same as the PCR prod. band size?

I upload my gel pic for u guys to help me analyse..thanks~ ^^

FACS ANALYSIS

Sat, 04 Jul 2009 03:40:14 -0700

Can nyone tell me wats d minimum cell number required for doing FACS analysis.

Guanidinium Chloride

Fri, 03 Jul 2009 12:55:33 -0700

Hi all, I am using a stripping protocol with Guanidinium Chloride to strip my PVDF membranes. It is a very simple protocol and it works at another workgroup in another lab just fine. The problem is that my PVDFs become all black after stripping, I dont get any signal, just background.
Has anybody had any experience with Guanidinium Chloride? Any ideas of what might be going wrong with my system?

Thank you all so much