PCR ...introduction - (Sep/11/2006 )

hi ,

i'm new to the field of PCR, i know the theory of this technique, but i have never try to run it alone....
i read many topics and posts here, many said that contamination may affect the end results of a pcr reaction...can anyone explain to me how this happens??

what role MgCl2 have ? necessary for Taq polymerase activity?

and another Qn...is it possible to add one reagent to thermocycler at an elevated temperature...i.e. adding Taq or the DNA insert?? is this what is called "HOT START"?

regards...

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When you prepare a PCR reaction, you always prepare a negative control, i. e. a reaction where you don't add template and this should give you no product. Sometimes, you can have template carryover and you will see an amplification and this is what you call contamination.

The amount of MgCl2 mostly is suggested by the Taq manufacturer but sometimes one needs to play with the amount to have a better amplification.

The HOT START Taq polymerase is an enzyme that turns active after a period at 95oC.

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thanx dnafactory for your fast reply...

correct me if i'm wrong: in a negative control, if amplification happened and a product on the gel,this means it was contaminated, this contamination came from bacteria or any cellular microbes

what about tap water, same thing?

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The contamination is DNA template that shouldn't be there. Most of the times it happens because you didn't change a tip while before pipetting another solution. Tap water can contain nuclease etc, therefore it is always reccomended to use nuclease free water.
Remember to use filter tips, because you could have some DNA in your pipet and you could put it in your reaction... This is another way of introducing a contamination...

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"Hot Start" is any process at which the reaction doesn't start before initial denaturation (mostly 2 minutes @ 95°C). This process is used to decrease aspecific product formation (primers have matched to your template DNA at the wrong place, this can happen at lowered temperatures).

There are severall posibilites for doing this:

1. Prepare your reaction mixtures on ice, start the thermocycles and put the tubes in when it's sufficiently hot (above 80°C).
2. Use of an enzyme that's inactivated because it's bound to an antibody at room temperature. Due to this bound antibody, taq is inactive, but during denaturation, the antibody is released and taq becomes active.
3. Use of a mutated enzyme that's inactive at room temperature. During initial denaturation (which mostly takes longer than 2 minutes, but depends on the specific enzyme you're using) it gets activated.

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thanx all,

another Qn, (be patient )some thermal cyclers require addition of mineral oil to samples and some don't...what is the role of mineral oil here?

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Strawberry, it is for the samples to stay in the bottom of the tube. with the temps close to boiling, otherwise the reaction components and water in the tubes just kind of ends up all over the walls and ceiling of the tube because of condensation, and the molecules don't have good access to each other for the reaction to proceed evenly.

if your thermalcycler has a heated lid, you don't have condensation trouble and the components pretty much stay in the bottom of the tube where they belong

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hi straberry
check this link
http://www.idtdna.com/techvault/technical....amp;position=11

this one has many answers for some questions about roll of every component of pcr reaction

http://www.idtdna.com/support/technical/Te...in_Reaction.pdf

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aimikins..thank u so much
let me ask few SILLY Qns: it's important for samples to stay at the bottoms to be near the required denaturation temperature...but could this also help preventing from evaporation!?

is mineral oil like other oils or has something special?

forgive me, but i have no experience..

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spanishflower...thanx alot, the sites are so useful ...appreciating your help

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