PCR - (Mar/26/2006 )
Hi,
Is there a particular order of pipetting the PCR reaction mixture? Water, then Pfu buffer, then DNA template, dNTP mix, primers then Pfu polymerase/
Thank you.
I do water, buffer, dNTPs, MgCl (or MgSO4), primers, polymerase. Mix thoroughly and aliquote out, then add DNA to each.
I make PCR mix of everything except primers.
The order of adding is
1. Water
2. MgCl2
3. Taq buffer without MgCl2
4. dNTPs
5. Pfu polymerase
6. Taq polymerase
7. DNA (if its same for all reactions)
I have read that enzymes don't stand high salt concentrations or even plain water. I don't know how certain that is. but with this order never had any troubles.
I follow the same when it comes to using restriction enzymes, add last after adding all salts and water.
Is there a particular order of pipetting the PCR reaction mixture? Water, then Pfu buffer, then DNA template, dNTP mix, primers then Pfu polymerase/
Thank you.
No.
Is there a particular order of pipetting the PCR reaction mixture? Water, then Pfu buffer, then DNA template, dNTP mix, primers then Pfu polymerase/
Thank you.
Accoding to some protocol, If you do the Hi-fi PCR, you'd better add the dNTP Pior to the Pfu which is robust in proofreading. for If you pippet the primers and Pfu before the dNTPS,your primers would be degraded by Pfu.
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the Finnzyme gives a recommended order of pipetting the PCR components:
ddH2O
10X buffer
Mg2+
dNTPS
Forward primer
Reverse primer
template DNA
DMSO(optional)
DNA polymerase
If you want to do a hotstart PCR ,you also could seperate the PCR components in Master mixtures, Template DNA ,dNTPS Primers, ddH2O in one tube. the other Tube contains 10X buffer and DNA polymerase. these two tubes contains same volume of rxn components, just before you start PCR , Mix these two Master mixtures.
Hi all...
Can anyone tell me when making working stock of Taq Polyemrase (conc-1u/uL)....do we add 10Xbuffer or is it made up juz it MilliQ....reply soon please...
Thankyou
It also depends what your aim is: If you have different DNA samples, what to try out different MG2+ concentrations, different primer try-outs with the same DNA template, do a hot start PCR manually (adding Taq later), you add the respective compound later directly to the tube. But I would almost always start with water, buffer and then as suggested by the others (I always add Taq at the end in a "normal" PCR). Too concentrated buffer is bad for enzymes.
Diluting Taq I would do in 1x buffer.
hi everybody
there's no specific order but we do:
water
pcr buffer
dntp's
forward primer
reverse primer
BSA or similar
Taq pol
and that's all folks!!
Usually it starts with water, buffer and last with taq. Other things can be added depending on which is the variable most of time which would be the template or primers. One of the reason is in setting up any reaction it is better to start with the component which is highrst in volume and least volume ones in the end. It is easier to add 1ul of enzyme to a liquid in the tube rather that directly on empty tube. Loss will be less and accuracy will be more.