Forgot to say, if you haven't already found it, this site http://www.gene-quantification.info/ has an absolute mass of information and links, I use it all the time :-)

Sana, I performed serial dilutions of cDNA for each of my primer sets, this gives you a standard curve that performs several functions:

1) Identifies the optimal cDNA dilution (aim to get a suitable Ct with all primers)
2) Enables you to calculate primer efficiency (not everyone uses this in their calculations but it's a good idea)
3) Provides you with a standard curve which you can use to determine a suitable threshold, if you wish.

I stopped using delta-delta-Ct and started using the REST program as I prefer it, horses for courses though, there's no one right way. If you're only using a single HKG it's not such an issue.

In terms of assessing the cause of poor reproducibility, have you checked your machine? I've found significant differences across plates, especially between the inner and outer wells on older machines using a solid heating block. If you want to assess this, make up a mastermix and cDNA solution sufficient for the entire plate, pipette it out and see what the well-to-well variation is like. Also, repeating any given experiment will give you a figure for the inter-assay deviation which is the other concern.

TJNZ, if you just feel that you don't really want to do a phd, then I'd be very wary of committing to one. As Bilbo says, it's hard work and you have to really want to be there. If you find yourself thinking of leaving science anyway then a phd may not be worth the hard grind. Don't worry yourself too much about letting the supervisors down, it would be far worse for you to commit and then leave halfway through because it really wasn't for you. While they may be initially disappointed they'll also be glad you were honest.

That said, if your discipline is any sort of lab or "straight" scientific research then I don't think a masters in sci com would help you if you decided to go back to the phd plan. A relevant masters will be an asset (although your work experience is clearly sufficient in terms of getting you accepted) but sci com courses, while they will teach you all kinds of media skills won't give you much you can apply to research. I was tempted by the course at Imperial myself but decided to hedge my bets in academia until I was sure, as it's a lot easier to leave than it is to get back in.

Thanks Nick,
It's a good point. The methods I'm using are fairly standard, however the specifics of them (how long temperatures are applied in PCR stages, the exact proportions of standard reagents used, and so on) are very variable, someone will have used the same as me at some point, but it wouldn't be worth tracking down. The tables just give a more user-friendly display of some fairly basic info.

Cheers Bilbo, I suspected as much (this is going to be one big table index!)

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This is probably painfully obvious to everyone else, but....

I'm using lots of tables within my methodology sections, listing reagents, mixes etc. Do I need to formally label them (Table 1.2 etc) and have them within the table lists? It's just that there are so many and they're so basic :s

ps - please excuse the spelling error in the title, I blame Mondays.

Today is likely to be low on words, I'm mosly doing analysis (which I thought I'd done before, but upon writing up it seems utterly inadequate) and making some figures. I feel rather like Alice, lost in a maze of genetic code and oversized diagrams. Luckily my cat is more cute than cheshire.

Whilst I agree with clogsy that you're fortunate to be kept on post-phd, I don't think that excuses your supervisor going back on his word. It doesn't sound as if this is dependent on your official qualifications, more your supervisor's own decision about what he can/wants to pay. Unless you have an official job title that corresponds to a specific pay scale at your institute, I don't imagine there's much you can do, and if you're only staying a couple of months I'm guessing it's all pretty informal? It's bad behaviour on the sups part but unless you have the original offer in writing (and are willing to fight him on it) then I imagine it's a case of "like it or leave".

Sadly true Eska, there are women out there who make me genuinely ashamed for my gender (hubby's psychotic Ex for one).

I'm afraid I have no experience of either, but I'd agree that a fellowship is not to be given up lightly. I take it it's not possible for you to visit Harvard before making a decision?
Aside from how friendly the place is, what were the supervisors like? This is so important in the course of your phd so I would really make it a priority. As Satchi says, most departments are pretty friendly and both these universities should be well set up for postgrads.

I wouldn't worry too much about getting less money (unless its so much less than it's going to seriously impact on your work or standard of living) but I would worry about the certainty of you getting the fellowship. It would be terrible if it fell through and you'd turned down Harvard.

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Set yourself small targets to start with, really small! I find that my motivation is primarily dependent on two things, confidence, and having made progress. When I'm working well the motivation just feeds off that, so my trick is just to find a way to get started and feel on top of it. Hence, small achievable goals that I can triumphantly tick off my list.

As Sneaks said, mytomatoes.com is great for this. Set yourself a target of just one or two tomatoes to start with, it's such a short space of time per tomato that it won't feel intimidating. As you get into it, raise the bar a little. Be realistic about what you can do whilst also working full time, as nothing that will sap your motivation faster than failing to meet your own targets.

In terms of writing itself, have you tried free writing just to get yourself going? Set yourself a time of ten minutes and write continuously for it, doesn't matter what on. Next, try doing it for your topic or current reading (no stopping to look at notes, this isn't going in the thesis). Again, it raises your confidence and stops writing feeling so overwhelming.

Oh, and rewards! Som people here give themselves a little gold sticker in their diary for every day they write. I schedule in activities and meetings with friends to look forward to. You could even promise yourself a little present for when you meet a certain goal.

I know what you mean sneaks, it's good for getting me started but tends to go off far too soon. I just ignore it, work through the break and then take a longer one after say two tomatoes (apart from anything I'm physically incapable of a five minute break, too short even to make tea!). Mainly I just like the encouragement of logging my progress so I can see that I'm getting somewhere.

I could do with that encouragement today, I'm so sleepy and the thesis just seems impenetrable.

Moomin, I held my breath as I went to open this post, hehe. I'm so pleased for you, CONGRATULATIONS DR MOOMIN!!!!!!!! Hope you're celebrating in style!

Hmm, drilling into the topic and questioning you will help you learn. But being entirely negative and just shooting you down is not constructive, I think some supervisors really don't understand (or don't care) about the basics of good management. I also think there can be an element of ganging up in these meetings, no one wants to look like the soft touch. Is there a member of the team who is more approachable? I wonder if you could go to them and explain that you're having serious doubts about your abilities and finding it hard to put their feedback into context, could they clarify what they expect at this stage? I'd also second wally's suggestions, when you get feedback feel free to engage with it and say "Ok, so how would you suggest I modify my approach?". It looks mature and will help you get something useful out of their criticism. Also, do they have other phd students? If so, it might be worth talking to them to see if their experience is similar, my guess is that your abilities are not the problem ;-)

Well done Bwad, keeping fingers crossed for you.