Colony PCR or PCR with Growing culture in Broth - Trying to detect the cloned fragment in living cells (Jan/18/2007 )

Hey Guys
Another Question for you.
What is the best technique to do PCR on living cells, cells from colony or can I directly take cell from overnight cultures which I use for plasmid extraction. I tried it once while taking 1 microliter from the broth culture in a total volume of 25 microliter. It worked and I got the amplification but I am not sure, will it work in every case.
Any Suggestions are highly appreciated.
And if any body has a nice protocol for this, I will request to post it here too.
Thanks in advance.

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if the cells are oldish (growing for >16hrs) I would avoid using them for PCR. This is true for both colonies and cultures.. cells growing more then 16hrs greatly increase their production of liposaccarides (slime!) which intefers with the PCR reaction.

Otherwise (provide sufficient dilution) and the cells are 'fresh-young' there is no difference between the two methods. Personally I prefer doing my PCR on colonies.. i use colony PCR for screening purposes and doing the screen on colonies saves me a day.

Use a 96 well plate to hold my colony dilutions.

Fill 96 well plate with 50ul sterile distilled water
Pick 1 isolated colony with pipette tip and place said colony into a well.
Repeat as many times as desired.

PCR mix per reaction (but this is usually made a single master mix)
4.22 ul sterile distilled water
1ul 2mM dNTP
1ul 10X taq buffer
0.5ul primer forward
0.5ul primer reverse
0.08ul Taq
0.2ul 50mM MgCl2

Volume 7.5ul

Add PCR mix into 96 Well PCR plate. Then add

2.5ul Colony solution

Final volume = 10ul

Cycling conditions

1 = 95 Celsius (4min)
2 = 94 Celsius (30 sec) melting
3 = Tm (30 sec) anealing
4 = 72 Celsius (1min) extention

Number of cycles 34.

Of course the above conditions can be changed. the time for each step is a little too long, but I don't bother to optimise it for each individual screen.

EDIT: The primer concentration is 10mM, made by diluting a master stock (100mM) with sterile distilled water.

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Thanks a Lot.

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Hi, I've been getting negative results all this time for my colony pcr screen! White colonies with negative results really depressing
My colonies have been on the plate for 2 days (unfortunately) cause they were really too small to be picked.
Would it also matter if I had resuspended the colonies in LB (5ul) instead of sterile water? Had realised you used 50ul!!
Maybe i should try out your dilution method as well.

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yes, I certainly agree. Do dilute your colony solution. If the colony solution is too concentrated, the PCR reaction will either fail or become very smeary.

If only a small amount of LB entered the PCR mix, 2ul or so, there should not be any effect. But personally I use sterile distilled water. Should I decide to keep the colonies in the 96 well plate for an extended time (>2 days), I later add LB to the wells.

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Hi perneseblue,
I just added another 50ul of distilled water as u suggested.
But unfortunately, still nothing turned up
Have tried on 36 samples !

Did u do any serial dilutions, or change the volumes of Colony dilutons for your PCR screen?
Anyway, I'll be repeating the TA cloning step once more since my plate is about 4days old now

Is there any other factors other than colony dilutions? I've browsed through the forum and it seems that there are also other members facing similar problems: picking up white colonies but not finding the inserts~
I'd like to foresee any upcoming problems and be mentally prepared~
Thanks!

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Hi
What primers are u using for the amplification. I have used 1 microliter of the broth culture for amplification and it worked well. To confirm the amplification with extracted plasmids and i get the same results.
How large is ur PCR reaction mixture? by the way. I think a 25 microliter reaction is enough.

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1. Add 100 uL water to desired number of wells in a PCR plate or tubes.
2. Toothpick colony into each well or add 10 uL of overnight culture
3. Heat in thermal cycler to 95C for 5 or 10 minutes. Heat blocks are not recommended, as the protocol does not work as well if the temperature is 93C rather than 95C.
4. Spin down the plate or tubes so that any cell debris is pelleted at the bottom of the tube/well. This removes the inhibitory components from the supernatant.
5. Carefully transfer 25 uL of supernatant to the PCR reaction, being careful not to disturb the bottom of the tube/well, even if you can't see any pellet.
6. PCR with forward and reverse to check for single/double inserts. Use vector only for a control.

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hi makosad05 and tfitzwater,

Thanks for your replies!
I use the same primers as my inserts, 40cycles
my PCR reaction is 10ul

I try this again

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The PCR product size. I find that under colony PCR conditions, Taq polymerase is reliably only for amplification of 900bp products and below. Anything larger, and the reaction's success becomes uncertain. If your product is on the large side, I would try reducing the extention temperature (to around 70 Celsius - 68 Celsius) compensating by increased extention time. (50% longer)

tm- what is the tm of your primers? Like any PCR reaction, all rules concerning primers design apply. There shouldn't be too large a difference between primers melting temperature.

The rule of the tumb for annealing tempearture is (tm - 5 Celsius). You can reduce the annealing temperature, a little further.

I would also consider testing more colonies, up to 72.

Lastly, there is there is the possibility that there the colony PCR isn't working because there aren't any colonies which are positive.

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