prabhubct

1) You could go to a grocery store or pharmacy and pick up a bottle of hydrogen peroxide -- the "home use" version is 3%.

2) If you want to dilute your stock, you need to dilute by  9.8 moles/liter divided by 0.88 moles/liter or by a factor of 11.1.

So, to make 1 liter, you need 1/11.1 liters of stock =  90 ml of stock, with the remainder, 910 ml, water.

Concentrated hydrogen peroxide is quite hazardous.  Be careful.

southtowns18, on 06 December 2012 - 10:22 AM, said:

BSA yes, FBS - no not really, the mothers are usually returned to the farm AFAIK, but the foetal calf is dead.

Quote

At least one animal died to make the  hybridoma, and these are usually propagated in FBS...  Ascites fluid can be harvested by an intraperitoneal line.  Polyclonals, the animals don't have to be sacrificed to make these, you can keep bleeding them, but it depends on the immunity raised and how long lasting it is.  It is probably kinder on the animal to sacrifice it.

southtowns18, on 06 December 2012 - 10:22 AM, said:

Why do you think sheeps are not killed for the meat?
Here its pretty normal to eat sheep.
But most sheeps are not killed in general because the wol is worth a lot more.
They just take some of the blood... but they do not need to kill the sheep for it.

Anyway, if you really want to know about this: contact the people where you buy the stuff.
Some companies have strict rules about this. Some will, for example, not work with animals being killed for just 1 product.

And in general: does an animal need to die? No!
For example horses are used to produce antidotes against snakevenom.. they dont kill the horse for it.

Also, http://en.wikipedia....al_bovine_serum, for you question about FBS en BSA

I have a question about animal-derived laboratory reagents and products. I’m working in a strictly in-vitro lab that focuses mainly on western blots and flow cytometry. I was wondering the cost to animal life associated with these reagents. These include:

FBS (fetal bovine serum) & BSA (bovine serum albumen): I figured these products are likely a byproduct of the slaughterhouse industry and therefore the animals are being slaughtered more for the meat than to get the blood to make these products. Am I right in thinking this?

Primary & secondary antibodies (raised in mouse, rabbit, goat, etc.): From what I understand there are three main types with differing degrees of cost to animal life

• In vitro Monoclonal antibodies: Once the hybridoma is created they can be grown indefinitely in vitro so there is minimal cost to animal life. Am I right in thinking this?
  
• In vivo (ascites) monoclonal antibodies: Does an animal have to die every time I place a new order for an antibody?
  
• Polyclonal antibodies: Again, does an animal have to die every time I place a new order for an antibody?

Sheep’s blood agar: Unlike the bovine products, I wouldn’t think sheep are being killed for the meat so maybe they are being primarily slaughtered to generate these lab products. Am I right for thinking this?

I’m asking these questions because I enjoy working in the lab but just want to know the cost associated with the products I use in regards to animal life. Also, can you think of any other animal-derived reagants/products that I left out that could be used in in-vitro experiments?

Thanks for any help you can provide.

Phenol denatures proteins.  Chloroform dissolves lipids.  I think you are on the right track with using PCIAA in your prep.  I would strongly recommend that you do a chloroform only extraction after the phenol, which will remove almost all of the phenol from your supernatent.  A precipitation and wash and you're set.

Curtis,

I can understand your frustration and the amount of time and energy you must have spent on getting the plasmid to work. But I think  you are being too harsh to her. Agreed, she should have replied by now,  but like leelee said, she might not just have seen your mail. She is just a caretaker of the plasmid, so its very likely, that she does not know much about the plasmid. She just sent you the sequence, she has on her record (which could have been wrong in the first place). So, she is not really to blame.

And why are you belittling your own work/ lab for such a small thing. You do not really know how things are in Oxford/ Cambridge, unless you are actually there.... (the grass looks greener on the other side)

Besides, if she really wanted you to have the wrong plasmid, she could have sent you one, why go through the whole rigmarole of sending you an carefully edited sequence. Or not send you one at all.

Yes, you have been at the receiving end of all this, and we really do not know what you have gone through, but at least give her one chance of clearing the air on this isssue. Questioning her education qualifications is a bit too harsh....  

I think if you do go ahead and complain you will be the one who ends up looking bad. Yes it is bad that she hasn't contacted you, but it could have been a honest mistake. She could be on holiday, maternity leave, moved to a new job, been having IT issues...

And in the end it will be your word against hers, and I think that people would generally believe it to be a mistake over malicious intent.

I know it is frustrating that you have wasted so much time, but chalk it up to experience and in future check everything. I think it is kind of unfair to imply that she isn't deserving of her job over what could very well have been a simple mistake.

Can I just say though, if it was on purpose, I don't think this is a common thing. We have often got reagents, plasmids, viruses,antibodies and mouse strains from other labs over years and have never once had a problem. There are good people out there

do you know why mops was selected in the first place? sometimes buffers are selected for reasons besides their pK.

if mops was selected only for its pK then you can use any buffer with the same or similar pK (i'm not at work so i don't have access to my list or i would make some suggestions).

Denny, on 06 November 2012 - 04:54 PM, said:

hey Denny...yup, it's a tough call but as you already said, if you keep quiet then you're probably risking the good reputation of your lab and your boss. They deserve to know the truth at the very least. I'm also thinking that you shld have just busted her in real time while she was making the mistake bec if you confront her much later, it might just degenerate into your word against hers.  As you mentioned, you already have a strained relationship and this is probably not a secret to people you work with so you might appear as someone only trying to create mischief or just aiming to discredit her for your own personal reasons.

Is there a way for her to fix the mistake (cos data from contaminated samples matter and if she knowingly provided contaminated samples to be assayed then that's a form of misconduct)  so you can avoid involving the higher-ups? But I would still try to talk with her first. I know it's a hard decision but if you ignore it or let it pass, then you are condoning her action or allowing her to think that she can get away with anything and in the end, you are just as guilty plus you wouldn't feel good about yourself either. Anyways, doing the right thing is never easy for anybody but hopefully we have the courage of our convictions.

The solubility of NaCl in H20 is about 360 g/l - you have 750 ml of water (therefore 360*0.75=270) and 292 g...add some more water.

Actually thinking things will 100% work this way is a first step to have a frustated life as s scientist. That's why they are called hypotesis, we don't know if they are true, we test them, if you got too excited about the fact they WILL work (instead of the fact it MAY work) you will naturally get frustrated and finally burned-out very quickly. Because there is no 100% success in any story, even less in any Science or Nature paper.

Get excited by the possibilities, not certainities, leave your mind open for for the fact, this one hypotesis won't be proven right, so you can immediatelly think of a new better one. Get excited by the fact you are uncovering the mystery, not by the fact you will get the one answer you want to hear. That's a science. Does it work? Excellent, so this hypotesis is true. Doesn't? Excellent, that means you got new piece of information you can use, new theory to make.

Other question alone are technical problems, you can't even prove anything, because you can't get the experimental system to work. Now that is frustrating, because it stands in the way of getting any knowledge. But in this way you come to analyse the problem, become the problemsolver, break it into pieces and work on each one to finally find the key, or just step aside and find incredibly simple solution out-of-the box. New source of excitement and contentment. Or sometimes you don't find any, you finaly need to give up and let go. But there are sure challenges that can't be answered now, and maybe they will be in the future.

You need to find a constant way to motivate yourself in this job. Because there sure are days of frustration, but you also need to have days when you're excited. And sometimes, when sou are so tired.. then the only thing that still takes you further is the restlessness and obsession, well.. that's the time to take a vacation

LOL...
Just a song for you

Duffy - Rain On Your Parade Lyrics
I wish you well
I hope you survive
I hope you live, oh baby, so I can watch you cry.

You are not alone... I'm doing my master now and my life sucks as well...

The very first thing I would do is have a look at your institute/university's pay regulations, and then the regulations of the state you are living in also. Is there some union or employee services that you can contact for help with this?
Find out exactly what your rights are so that you have the information to back you up.

Then I would have another (non-emotional, non-angry) chat with your employer about your concerns, be firm but not agressive. Ask for what you want and then give him/her time to think about it. Have evidence for why you think you are worth this pay rise (ie experience, work successes, work ethic etc), as well as evidence for why it is unfair to continue to pay you less (ie regulations, people with less qual doing same job for more etc). Don't demand an answer straight away. Try not to play the blame game.

You don't say what country you are in, but is there some kind of organisation that deals with employee rights etc that you could talk to? You may need to take it further and they could advise you as to the best avenues to go down.

Of course, if the pay is within the rules and regulations- and there is no chance of a raise, then you need to have a good think about what to do next. If your resentment and bitterness is going to build, you will end up miserable and you don't want to be in a job that you hate going to everyday. You may need to look elsewhere.

And most of all try not to take it personally!

Edit: oh sorry, I see you are from the US in your info now , I don't know much about pay scales and employee rights in the US, but I wish you good luck in getting what you feel you deserve!

SF_HK, on 09 October 2012 - 01:50 AM, said:

150ml water

My quick summary (not applicable to ultracentrifuges!):

It doesn't much matter except for breaking things.  2000-3000g for 96 well plates.  6000g or so for thin wall pcr tubes (you will be unhappy when your precious sample goes into the rotor).  8000g for most conical 15 and 50 ml tubes.  15000g for eppendorfs, including column minipreps.  Final elution of miniprep columns into capped tubes can be done at 6000g if you want to keep the loose caps on the tubes.