Protocole for Q-PCR
Material :
· Platinum Taq-DNA polymerase
(INVITROGEN, 500 reaction, 10966-034)
5 U/ml
10 X PCR buffer (200 mM Tris.HCl, pH 8.4; 500 mM KCl)
MgCl2 50 mM
· dNTP
DNA template preparation :
[Plasmide] =
[Template] =
Master Mix :
[C]final / 1 reaction (ml)DNA template / 10
10 X PCR buffer / 5
MgCl2 / 3 mM / 4
dNTP (25 mM) / 1
Primer mix / 1/40 / 1
Platinum Taq / 30 U/ml / 0.3
SYBR green dilution / 5
H2O qsp / 23.7
50
SYBR green titration
SYBR green :
From ….
In DMSO (DMSO is an inhibitor of the PCR reaction)
Aliquot by 10 ml
Conserved @ -20°C
First dilution : SYBR green 1:1000
1 ml SYBR green in 999 ml H2O
Dilution :
Take care of the dilution in the master mix : 1:10
Dilution / ml 1:1000 / ml H2O / Final volume (ml)1:10 000 / No dilution
1:20 000 / 50 / 50 / 100
1:30 000 / 30 / 70 / 90
1:40 000 / 25 / 75 / 100
1:50 000 / 20 / 80 / 100
1:60 000 / 15 / 75 / 90
1:70 000 / 10 / 60 / 70
1:80 000 / 12.5 / 87.5 / 100
1:100 000 / 10 / 90 / 100
Run a Q-PCR to determine which dilution of SYBR green give the best fluorescent efficiency.
Use this dilution to determine the good MgCl2 concentration for the reaction.
MgCl2 concentration determination
Prepare the MgCl2 dilution :
[MgCl2] in mM / ml MgCl2 / ml H2O / Qte MgCl21.5 / 1.5 / 8.5 / 75 nmol
2.0 / 2.0 / 8.0 / 100 nmol
2.5 / 2.5 / 7.5 / 125 nmol
3.0 / 3.0 / 7.0 / 150 nmol
3.5 / 3.5 / 6.5 / 175 nmol
4.0 / 4.0 / 6.0 / 200 nmol
4.5 / 4.5 / 5.5 / 225 nmol
5.0 / 5.0 / 5.0 / 250 nmol
Volume final 10 ml
Prepare a master mix with MgCl2 dilution
1 reaction (ml)DNA template / 10
10 X PCR buffer / 5
dNTP (25 mM) / 1
Primer mix / 1
Platinum Taq / 0.3
SYBR green dilution / 5
H2O qsp / 17.7
MgCl2 dilution / 10
50