Thread: PCR on a bacterial pellet

Hi there,

I am performing a PCR-reaction on a bacterial pellet and getting no results. The pellet was freeze-dried en then resuspended in 500 microliters of LB-medium.
Is it a problem that i'm performing it on the pellet? I think it doesn't matter because the cells will burst open at 95 degrees en the gDNA will be free in the PCR mix.

this is my PCR mix:

Cell Culture	2 (10x diluted)	1	1.5	2
FWD primer (10 µM)	5	5	5	5
RVD primer (10 µM)	5	5	5	5
2 mM dNTP’s	5	5	5	5
10x pfu pol buffer	5	5	5	5
10xdiluted pfu	1.25	1.25	1.25	1.25
ddH20	26.75	27.75	27.25	26.75
Totale	50	50	50	50

And my PCR program is:

5 minutes - 95 degrees
30 sec- 95 degrees 30x
30-60 sec - 50-58 degrees 30x
7 min- 72 degrees 30x
10 min - 72 degrees

After a agarose-gel is see the gDNA (i think) in the slots and a smear in the lanes but no bands. I also have a positive control so the components ( dNTP's, POLymerase buffer, polymerase) are working.

Does someone have a solution for this problem? Or need i to do a DNA extraction because the cells do not lyse fast enough

And my PCR program is:

5 minutes - 95 degrees
30 sec- 95 degrees 30x
30-60 sec - 50-58 degrees 30x
7 min- 72 degrees 30x
10 min - 72 degrees

Wrong..

Try another 30 seconds with that one.
Try 30 secs with that one, the 30-60 strat works sometimes to increase the efficiency, but if it fails totally, skip it.

Is it a problem that i'm performing it on the pellet? I think it doesn't matter because the cells will burst open at 95 degrees en the gDNA will be free in the PCR mix.

Yes, that is a problem. Using that on your pellet will leave a lot of junk lieing around. First lyse the cells manually, and extract the DNA. Don't just fool around.

Furthermore you neglected to investigate the part your primers should target. What is it, how long is it, what does it do, how many C=G's or A=T's, how prevalent are they, and does it even happen in your bacteria?

Aaaand..

Last but not least. I don't see polymerase... If it is dilluted, you might want to check if it still works. Usually polymerase is very sensitive to buffer concentrations.

So.. i am not surprised you failed the experiment..

@zwolver

The step from 7 minutes to 30 seconds cannot be done, because the PFU polymerase has a amplification rate 500aa/minute.

The polymerase works because the positive control gives a positive result.

But thanx for your comment, i will isolate the DNA and fingers crossed that it gives a positive result

sorry i was a tad sarcastic, i am not familiar with PFU polymerase, i actually only use taq.. Not sure what the difference is there, seems you use a lot more of it, or it's indeed dilluted like i thought.

If the DNA replication wasn't finished, this isn't a great bummer. It'll finish it later.

In my idea all the 7 minut step does, is kill your polymerase.. Increase the number of cycles (35x or even 40x), and shorten the 7 minut step..

Check for other compounds that can help your polymerisation, as sometimes the dna doesn't melt, primers won't bind, or loops will form, or the primers react with themselves.. (primer dimers)

It is possible to do PCR on whole cells; just lyse them first in ddH20 at 95C for a few minutes.

Since you state that you are seeing a large smear of DNA on your gel then really there could any number of things going wrong. As a first step in identifying what the problem is I would try reducing the concentration of sample DNA and see what happens. It's also possible that your polymerase may have degraded your primers if you weren't careful.

Yeah, i know you can PCR on whole cells, but i wouldn't start with that. If your scraping your method you might try it, but not when your beginning.

the pfu polymerase doesn't get killed, i know that because of the positive control. That one gives fine results.
I found a different sequence of the gene of interrest in NCBI then i first looked up and made primers for. So maybe that is the problem. i have made new primers so i hope this will work. Also im going to do a PCR with 2.5 times higher primer concentration because of the pfu polymerase has 3 tot 5 exonuclease activity so maybe it breaks down the primers when they are annealed.

Originally Posted by ssgoj

Also im going to do a PCR with 2.5 times higher primer concentration because of the pfu polymerase has 3 tot 5 exonuclease activity so maybe it breaks down the primers when they are annealed.

Usually increasing primer concentration doesn't increase results. You simply need sufficiënt *2. If your concentration of primers it to high, it rather binds to itself then to your target DNA.

I have to read up on PFU though..

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