almost a doctor

To dilute your cells from 1.2x10^6cell/ml -> down to 0.5cell/100ul you'll need to do a several 1:100 (or 1:1000) dilutions first.   Here is how I'll do it.

1.2x10^6cells/ml =  1.2x10^5cell/100ul (this way you have your stock and your desired final concentration in the same units).

Now, you could use C1V1=C2V2  but this results in having to take x10^-5 microliters which is pretty much impossible.  So, here is where the several 1:100 or 1:1000 dilutions come in.

1.  dilute your cells 1:1000 ->   now you have a 1.2x10^2 cells/100ul  
2.  dilute this new stock 1:100 ->  now you have a stock at 1.2cells/100ul  

Now you can apply C1V1=C2V2   assuming you only need one plate and allowing for a bit extra I'll consider V2=10ml.  C2=0.5cell/100ul and C1=1.2cell/100ul I'll let you do the rest   

remember to prepare enough volume of your intermediate dilutions so you have enough V1 for this!!  

Hoe this helps.

Can you tell us what you've done, and why you've concluded the primers don't work?  

Also, are this newly designed primers (ie. never tested) or have they been shown to work before?

The more you can tell us (detailed protocols you've used), the better we can help you.

I have a few questions and comments to try understand what's going on, and try to help you.

Did your b-actin looked the same as in this picture the time that "worked"?  I mean, you seem to have a lot of primer dimer (bright bands at the bottom of gel) compared to what I assume is your specific product (a lot lot fainter).  I would not call that a "working" PCR much less would I use it as standarised.  
Also, did your other genes show the bands at the bottom in the PCRs that worked? Again, if they did I would not consider that a working reaction.

If your reactions looked like this before I think they need more optimisation to call them "standarised".  If, on the other hand, they did work fine (no primer dimer), I'll say you either have a problem with your template or some of your reagents as bigudukaz suggested.

A few things you can play with aside from annealing temp to optimise your reaction are:

• Primer concentration:  I think it needs to be lower, in view of how much primer dimer you have.  If your previous "working PCR" did not show this primer dimer, this might not be the issue.
  
• Template concentration (what is your template? is it stable?).   You are either using too little template, and if it is not very stable this time the primer dimers have "win", or you are using too much template and it has some inhibitors that are messing with your reaction. Should be easy enough to try a reaction with more template, and another one with less (dilute your template so the possible inhibitors are diluted too). For PCR usually less is more, if you need tone of template for it to work, is not really working.
  
• MgCl2, DMSO.... varying concentrations of this can help get rid of primer dimer an favour your specific product.  I don't know how easy this will be if using a ready PCR mix, but should be doable.

Hope this helps.

What's your lysis buffer?

Could it be that your protein concentration is high and some of it has come out of solution and precipitated? This are usually hard to get back into solution.

Just a thought

Peniel, on 04 March 2013 - 07:18 AM, said:

Are you sure those amounts are right?  I mean... 1200mg = 1.2grams sounds like a lot for invitro assays!!   Furthermore, if you are talking in mg the molecular mass of the drug is irrelevant, as is the volume you might use.  

If your amounts are the correct ones, and the drug comes in 10mg and 50mg, yes you are going to have to buy loads.... 75+150+300+600+1000+1200=3325mg... and that will be just for one day for one well!  (if I've understood your post correctly that is).