Trying to lyse cells to get PCR template - Need something simpler than a miniprep (Feb/01/2007 )

My apologies if this in the wrong forum; it's quite a broad problem.

I'm struggling to figure out how to screen transformed algae (Chlamydomonas) cells for my insert using PCR. A standard colony-PCR (eg picking with toothpick) doesn't work like it does for bacteria, probably because of one or more of these reasons:
-not enough DNA. Chlamydomonas colonies probably aren't as dense as bacteria, or
-cells aren't lysing in PCR reaction, or
-cellular matter interferes with Taq

So far I've tried larger amounts of algae (a loopful rather than a toothpick), tried boiling in an EDTA solution + vortexing for 10 minutes before taking a sample as template, I've tried boiling in TE + Triton X-100 and vortexing, and I've tried freeze/thaw in liquid nitrogen followed by sonication (in a sonic cleaner, is this not powerful enough? do I need a proper ultrasonicator?)

So far nothing has worked - right now I'm just trying to amplify a known gene as a positive control, not screening colonies yet.
In all this I haven't tried changing my PCR protocol, which is a pretty much standard protocol, except I increased the amount of MgCl to compensate for the EDTA. This made no difference.

I don't expect any of you to have any experience with algae, but what do you think might be the problem: not enough template (eg cells not lysing), or am I putting too much crap into the PCR and interfering with Taq? Note that some of my PCR reaction tubes have been green from chlorophyll.

Finally, the obvious question, why don't I miniprep? Because I have over 400 colonies to screen from 16 transformation events. I want to streamline the protocol and use microtitre plates.

Any ideas or suggestions would be most welcome!

so why the PCR should actually be positive? you say this is screening. maybe you just have negative results.

The idea is to use this for screening, but at the moment I'm just trying to amplify a known gene from the genome in order to get the protocol to work.

I just noticed something on my last gel - at the top of the gel where the wells are, there is a HUGE amout of DNA in the lanes from the Triton-X samples. I've never seen so much DNA. It must be genomic. So I think Triton-X is effective at lysing the cells, and I have lysed way to many cells.

I now think that my PCR is being inhibited by the cell matter. Perhaps I should use sodium-acetate to precipitate the proteins? And then take some of the supernatant. Also, I will try adding a previously-prepared miniprep as a template (this is known to work, I have been using it as my positive control) and see if that gets inhibited by my cell lysate.

More questions:
-what sort of things do you know inhibit PCR? I know that SDS does.
-The last steps of a miniprep are to wash the DNA, by precipitating it with ethanol. What does this step actually wash away?

umm... Sodium acetate percipitate DNA. And some proteins too.

the ethanol percipitates DNA and RNA (and some proteins.. usually those bound to DNA). Other things like lipids and salts remain suspended. WHen the ethanol supernatant is removed, so goes these suspended material.

I know PCR doesn't like polysaccharides and liposaccharides. Maybe due to physical problems.

Yeah, I just discovered sodium acetate precipitates DNA
There's a protocol for this floating around the internet but I haven't managed to get it to work, and my colleagues say it can't be done. But I'm sure it can!

I suspect I'm using too much cellular matter as template. I know if you use too much bacterial cell matter it can screw up the PCR - probably due to saccharides in the cell membranes?
Thanks for your help!

We now suspect that the starch content of the algae is inhibiting the PCR. We're going to try this "agarose cleanup" method -

"Efficient removal of PCR inhibitors using agarose-embedded DNA preparations"
D Moreira
Nucleic Acids Research, Vol 26, Issue 13 3309-3310

Seems pretty good. Basically the cell lysate is mixed with liquid agarose and allowed to solidify. The agarose lump is then washed in TE for a few hours, removing a lot of contaminants but leaving the DNA trapped in the agarose. A slice of agarose is then used in the PCR.

I was going to suggest that sugars/polysaccharides might be inhibiting, having done some work with plants... I would suggest that going for a CTAB based protocol is probably the best way around that, but very time consuming and not what you are looking for. There is a plant DNA extraction kit from Qiagen which is pretty quick and gives good results.

Yes, we have that plant DNA extraction kit but it's out of columns, and I was hoping for a protocol that's easier and doesn't require buying another Qiagen kit - although we could wash some columns in HCl, apparently

Washing in agarose did not work, but in the end our protocol was very different to the Moreira one that I posted above - specifically we lysed with 10% Triton and didn't have any Proteinase K handy so we went without. We'll try it again when we track down some Proteinase K.

Also: remember I said that cells lysed with Triton-X and run on a gel show up with a strong fluorescence, indicating genomic DNA? It turns out that Triton-X is fluorescent.

Next method: Trizol extraction! For DNA! It can be done, correct? Either that, or Extract-N-Amp kit.

Yes, we have that plant DNA extraction kit but it's out of columns, and I was hoping for a protocol that's easier and doesn't require buying another Qiagen kit - although we could wash some columns in HCl, apparently

Washing in agarose did not work, but in the end our protocol was very different to the Moreira one that I posted above - specifically we lysed with 10% Triton and didn't have any Proteinase K handy so we went without. We'll try it again when we track down some Proteinase K.

Also: remember I said that cells lysed with Triton-X and run on a gel show up with a strong fluorescence, indicating genomic DNA? It turns out that Triton-X is fluorescent.

Next method: Trizol extraction! For DNA! It can be done, correct? Either that, or Extract-N-Amp kit.