11 replies to this topic

[GNANA]

Hi everyone...,

     I am not new here, now i am here just to proceed with confidence on my work...i got to PCR some of the exons and sequence it for mutation analysis...could anyone suggest me a good master mix (higher fidelity and at the same time works better without demanding much time for optimisation)  from your experience....
also suggest me a DNA clean up kit....
thanks in advance....

Gnana....,

Edited by GNANA, 11 April 2011 - 12:14 PM.

[phage434]

My go-to master mix is Invitrogen Platinum High Fidelity master mix, which is a a Taq-Pfu mix.  Also available in a hot start version, which I don't find necessary.

[GNANA]

thank you..,i have accuprime Pfx (invitrogen), but it works for some DNA and not in others, the DNA are older and also needs some purification..., whats ur opinion about accuprime pfx....

it would also helpful if someone comes out with their experienced mastermix for mutation analysis...

[Trof]

The best mix we ever had was QIAGEN HotStarTaq Master mix. I practically don't optimise anymore. It's hot start, no pipeting on ice needed. Even when ABI Gold mixes failed, this just worked. It's not a hi-fi enzyme, but we sequence everything with it on regular basis (more than 5 years) and never had a false call. Only that one template with a long (more than 10) polyX nucleotides caused the enzyme to slip, but that must be avoided by sequencing from the other side anyway.

[GNANA]

thanks a lot trof ....i go with your suggestion...

Gnana....,

[Trof]

Just to say, we only have DNA of good quality, from blood or cells. Izolated by phenol/chlorophorm and stored in 10mM Tris pH8. If you have issues with PCR inhibitors and unclean or degraded samples that may be the main problem, not selection of polymerase.

[Adrian K]

phage434, on 11 April 2011 - 03:08 PM, said:

My go-to master mix is Invitrogen Platinum High Fidelity master mix, which is a a Taq-Pfu mix.  Also available in a hot start version, which I don't find necessary.

Trof, on 12 April 2011 - 12:00 AM, said:

The best mix we ever had was QIAGEN HotStarTaq Master mix. I practically don't optimise anymore. It's hot start, no pipeting on ice needed. Even when ABI Gold mixes failed, this just worked. It's not a hi-fi enzyme, but we sequence everything with it on regular basis (more than 5 years) and never had a false call. Only that one template with a long (more than 10) polyX nucleotides caused the enzyme to slip, but that must be avoided by sequencing from the other side anyway.

wow... you people are super rich...
I still make my own master mix...

[Trof]

adrian kohsf, on 13 April 2011 - 03:20 AM, said:

wow... you people are super rich...
I still make my own master mix...

Well I do like 10 different genes a year, each has several exons, on just a small bunch of samples. If you need to optimise each and every single amplicon for just like 10 samples, you probably spent more money and more importantly time, than if you just set the first reaction and it works. If you have lot of samples for single assay, the situation may be different.
It was me who proposed using this mastermix in this lab, before that everyone was on cheap Taq (and troublesome Pfu mixes for sequencing) setting the reactions on ice (I hate to work on ice, you can possibly contaminate everything with the melted water) and still getting nonspecifities and things like that. And before they were using 50ul reactions, I started with 20ul, that saves some money too, and for a sequencing it's completely sufficient.
Sometimes expensive is not that expensive in the end.

[GNANA]

thanks trof.., first i purify the DNAs and then proceed with the PCR....thanks again for your concern........,,

Gnana......,

[Adrian K]

Trof, on 13 April 2011 - 09:03 AM, said:

Well I do like 10 different genes a year, each has several exons, on just a small bunch of samples. If you need to optimise each and every single amplicon for just like 10 samples, you probably spent more money and more importantly time, than if you just set the first reaction and it works. If you have lot of samples for single assay, the situation may be different.
It was me who proposed using this mastermix in this lab, before that everyone was on cheap Taq (and troublesome Pfu mixes for sequencing) setting the reactions on ice (I hate to work on ice, you can possibly contaminate everything with the melted water) and still getting nonspecifities and things like that. And before they were using 50ul reactions, I started with 20ul, that saves some money too, and for a sequencing it's completely sufficient.
Sometimes expensive is not that expensive in the end.

Any vacancies for a short internship in your lab? I'm interested to join you and learn from you.

[phage434]

There is a huge advantage to using a standard mix of reagents when you are trying to optimize many things simultaneously.  If you mix PCR with buffer, water, enzyme(s), dNTPs, primers, and template separately, you have many chances of things being contaminated, out of date, or lost.  Standardizing everything except primers and template makes things much easier and consistent.  You can do this yourself, of course.

I also basically don't optimize.  Annealing is always at 55C, extension at 68C.  The extension time is varied.  This almost always works with well designed primers.  If it doesn't, then I sometimes try a gradient PCR -- once.  If that doesn't work I redesign primers, which almost always solves the problem.

I've never found it necessary to make up PCR reactions on ice, although I can see the theoretical reason why it might be necessary.

[Trof]

Since I was cloning last week I'd just note, that HotStarTaq is great on normal applications and direct sequencing of PCR products, but I wouldn't recommend it for PCR used for cloning. I had 1600 bp product that was faint, so I used 33 cycles (quite a lot, I know) and three out of four plasmids have at least one mutation.