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Homemade TaqMan PCR Mastermix? - (Apr/01/2007 )


I've just registered to this forum and first of all I want to greet the community smile.gif

and here comes my first question: laugh.gif

currently, i'm doing TaqMan real time PCR with an Applied Biosystems PCR mastermix which is very expensive. so i'm trying to make some by myself which would cut the overall costs of one rxn down to half.

For my self made mastermix i use the Hotfire polymerase from Solis Biodyne. This is a hotstart enzyme with 5' to 3' exonuclease activity.

i have already done some optimisation steps but i still have some problems regarding PCR efficiency.

i'm always doing standard curves of 6 serial dilutions with 4 replicates with the HPRT housekeeping gene and compare the ABI mastermix with my selfmade one (always 20µl rxn volume). i'm using cycling conditions recommended by the manufacturer of the HotfirePol:

15min 95°C
and 40 cycles of
15s 95°C
30s 60°C
30s 72°C

Interestingly, also the Applied Biosystems Master Mix performs very well at these conditions.

here are the curve-equations:

1. attempt:

y = -3.3332x + 29.404; R² = 0.9966; Efficiency = 99.5%

Hotfire Pol (1U polymerase with 2,5mM MgCl2):
y= -3.9761x + 34.985; R² = 0.9714; Efficiency = 78.4%

Comments: So, the ABI R² value and efficiency look very fine, Hotfire Pol is quite poor.

2. attempt:

y = -3.4696x + 29.502; R² = 0.9973; Efficiency = 94.2%

Hotfire Pol (0.8U polymerase with 3.5mM MgCl2):
y = -3.75x + 32.352; R² = 0,9892; Efficiency = 84.8%

Comments: ABI quite consistent, Hotfire Pol values have improved

3. attempt:

y = -3.436x + 30.803, R² = 0.9974; Efficiency = 95.4%

Hotfire Pol (1U polymerase, 3.5mM MgCl2, 0.4µl ROX dye/rxn)
y = -3.6346x + 32.277; R² = 0.9941; Efficiency = 88.4%

Comments: ABI again very good, Hotfire Pol R² value has significantly improved after adding ROX dye (passive reference) to the mastermix, Efficiency has improved by 10% compared to 2.5mM MgCl2.

here is my self made master mix receipt (latest version) for one rxn:

2.0µl 10X buffer (w/o MgCl2, Solis Biodyne)
0.4µl dNTPs (10mM)
2.8µl MgCl2 (25mM)
0.4µl ROX dye (Invitrogen)
1.0µl HPRT primer/probe mix (Applied Biosystems)
0.2µl Hotfire Pol (5U/µl, Solis Biodyne)
8.2µl water
5.0µl cDNA

so, do you have any ideas how i could boost the PCR efficiency for another 10%? wacko.gif
More enzyme?
More MgCl2?
Less dNTPs?
... ??

Any help would be appreciated and sorry for that long post.....

-Ned Land-

Try this buffer composition. it work for me.

500 μM of each dNTP,
50 mM Tris·Cl (pH 8.3),
3 mM MgCl2,
75 mM KCl,
and 10 mM DTT.