leelee

Depends on you and how you work best. I work best under pressure and I need external deadlines to keep me motivated (I suck at self motivation... perhaps science isn't for me after all??!!...).

I WISH my supervisor had been more "pushy" with me early on, as I feel like I am quite behind and have wasted time faffing about with things that someone should have told me were time wasted. I needed more direction. But, hey, at least I learnt something, right??

The only time I think "pushy" can be bad, is when it is coupled with micro management. It is one thing to expect results and to guide your staff and students towards them, whilst still allowing them autonomy. Quite another to be over their shoulder, second guessing every move they make!

I suspect that the main reason the blog suggested using it only for transformation is that you will then have a glycerol stock of bacteria containing the plasmid that you can go back to time and again to produce more plasmid if and when you need it.

Rather than using it all up for PCR and then having to send for more when you run out. Or contaminating your single aliquot while using it for multiple PCRs etc.

I can't see any reason why you wouldn't be able to use it for PCR. I routinely run QPCR on DNA samples in saliva collected onto filter paper. I actually just put a little punched circle of the paper directly into my master mix and run. There are specific tools available for this purpose, if you need to be doing a high number of these.

If I were you, I would elute into water then use a few ul to transform into bacteria and a few ul for your PCR, then store the rest as a back up until you have verified your transformation was successful and you have isolated and verified the plasmid from your new stock.

Actually, if you are talking about a ratio, then a two fold dilution is 1:1
That is, one part bacteria to 1 part diluent.
1:2 means 1 part bacteria to 2 parts diluent (or a 3 fold dilution).

Often scientists use this ratio notation incorrectly, so quite often, people will still know what you mean, but I can't help myself from pointing out it is wrong..... I can be a know-it-all sometimes   

Every virus is different. They all target different receptors and are internalised by different mechanisms (or multiple mechanisms). Perhaps if you give more specific details, someone can help you?

I agree that teaching is a brilliant way of learning! You have to really understand the topic/technique/idea to be able to teach it to others.

Try and synthesise your reading into some kind of presentation- say for journal club in your lab or department. Even if you don't end up presenting it. It will make you focus on the important stuff and really read to understand.

Also, there is ultimately NO shortcut to learning. You can lament that reviews are long, and you want answers quickly, from search engines. But the fact is that quality, in depth knowledge (the kind you need for a career in science) cannot be found in jazzy, summarised bite-size pieces.

My advice is to keep up with your reading. But also talk to people. Watch how they perform their experiments. Ask them why they are doing it such-and-such a way. Have regular chats with your lab mates and supervisors about your field. Don't be afraid of not knowing much, no one expects you to be an expert straight away and it will come, I promise.

SDS can precipitate out of solutions at RT, so if buffers that do contain SDS have a whitish precipitate in them, some people can mistake if for contamination.

http://lmgtfy.com/?q=rt+pcr+vs+pcr

Alert: Thread hijack ahead  

Curtis, on 09 January 2013 - 08:24 PM, said:

Actually, both are acceptable. Usually one would use lower case, but litre is the exception, where upper case can be used when concerned about mixing up a 1 and an l. And also, most countries spell it "litre". I'm pretty sure it's is just the Americans who use "liter".

(source: Internationional bureau for weights and measurements http://www.bipm.org/...apter5/5-1.html)

prabhubct, on 30 November 2012 - 09:16 AM, said:

I agree. You'd first have to prove that she deliberately sent you the wrong sequence. Which would be very difficult. Particularly as the error is some repeats, which seems to me fairly easily explained as a copy/paste type error.

So the worst you could prove is that she ignored your emails. Yes it is rude and annoying for you- but I hardly think it makes her a bad scientist or bad at her job or whatever- and I really doubt her boss or institution will care.
If you take it further, best case scenario, they believe you- then what? You'll get an apology. That's it. I really think you would come out of this worse than she will! And you could kiss goodbye any chance of getting anything from them in the future.

Honestly, it is not unusual for communication with people from other labs to take some time. They are busy, they are doing you a favour and you are not their priority.

I understand your frustration, I really do, but I think you have to let this one go. You don't want to harm your own reputation for something like this.

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

You can use either.

Are you isolating from tissue culture supernatant? Or a sample of some kind?
I probably wouldn't shell out for a kit if you are using it for PCR, unless you think you'll need really pure DNA.

I think the variability might depend on your sequence (as each nucleotide is slightly different)?

Try this Invitrogen page

I've done what you just described (pellet, aspirate media, freeze), and the subsequent mini-prep worked just fine.

Hmmm. Ok. What about increasing the centrifugation time to 10 min?

Also, I have found that centrifuge speeds that I use for my big tubes (15 and 50ml) in the larger benchtop centrifuges pellet my cells MUCH better than equivalent rcf values in a microfuge. I don't know why this is (350xg for both).

I get around it by increasing the speed for the microfuge- but by this stage my cells are usually fixed and so I don't have to worry about viability.

Hopefully someone else has some tips for you

It will be completely fine. It is actually a good way to store DNA long term.