[urgent help] Band missing in PCR - (Mar/02/2006 )

Hi, all....
I had a problem with my PCR and no bands were found ....

This was what I did...

1) Overnight enrichment broth culture

2) obtained 1ml

3) centrifuge (discard supernatant)

4) pellet

5) resuspend pellet in ultra pure water

6) extract DNA inside the cells by 100 C water bath

7) centrifuge

8) transfer supernatant to PCR tubes + add reagents

9) PCR


but to my surprise the result from the same set, same amount of sample under the same condition was not reproducable...

Some wells had band while some don't...

what could have been the error to this?

I suspected that the pellet was accidently removed when I carried on step (3) by using a pipette to remove all the supernatant (p.s the pellet was too small to be visible)
if true, how can I avoid this error?

Many thx, I am really looking forward to advices and suggestions...

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try using a Midi-Prep kit...

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Can you explain a bit more about midi-prep kit?

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I don't know if hsp was going here or not, but I think you need a bit higher density of starting cells for your template. what type of cells do you grow? I have PCR'd with both S aureus and E coli with approximately the same method...except that I use enough cells to see a distinct pellet, and I also add steps to ensure lysis of the cells if I am using the gram-positives.

could you please give us more details? how much water do you use to resuspend? and, when I have done this, I have not performed a centrifugation (your 7th step)...is that necessary? since the DNA will still be bound up with the proteins with such a rough prep, then perhaps you are spinning it into the pellet at that step and lowering your yield?

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I was working with vibrio parahaemolyticus

I used autoclaved ultra pure water of 10.5 microliter to resuspend the pellet so that when I add up all my primers, it will be a 1:1 proportion to my 2x master mix.

The intense of the centrifugation step was to centrifuge the cell debris in the tube so that the only thing in the supernatant will be DNA.

Actually the biggest problem I am facing is that my supervisor wants the sensitivity of the PCR assay, which I think might not been neccessary, so I was asked to reduce cells/ml until I reach a point where no band was observed. In this case I can't use a observable pellet (as the cell/ml is too high)

Also, it might be true that some DNA had been spun into the pellet and is worth a try

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Are you trying to amplify a target on chromosomal DNA or a plasmid? In either event, I would get purified template (either a plasmid prep or a chromosomal prep), estimate the DNA concentration in your starting sample, and do PCR on dilutions from that.

Theoretically (and depending on your PCR protocol), a single cell would be enough to allow amplification, so I'm not sure how you get to "the sensitivity of PCR".

It is very hard to control (and get consistant results) from whole-cell PCRs.

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