5 replies to this topic

[Biocat]

Hi all,

Recently I have problem with PCR amplification.
First i used High fidelity PCR master (Roche) to amply gene from genomic DNA, but failed when i tried to change different parameters (annealing temperature, different pirmers etc). When i used Taq enzyme do again, it works, i can see very nice band on gel.
Because of considering relatively high mutation of Taq, i tried to use pfu polymerase again (followed by mannual), but didn't work either.
So i wanner to know what's main points which differs results so much.


Biocat

[ProteinWork]

Biocat, on Apr 19 2010, 10:42 AM, said:

Hi all,

Recently I have problem with PCR amplification.
First i used High fidelity PCR master (Roche) to amply gene from genomic DNA, but failed when i tried to change different parameters (annealing temperature, different pirmers etc). When i used Taq enzyme do again, it works, i can see very nice band on gel.
Because of considering relatively high mutation of Taq, i tried to use pfu polymerase again (followed by mannual), but didn't work either.
So i wanner to know what's main points which differs results so much.


Biocat

Use a new batch of enzyme, or increase the amount of enzyme you put in.

[swanny]

ProteinWork, on Apr 20 2010, 01:06 AM, said:

Biocat, on Apr 19 2010, 10:42 AM, said:

Hi all,

Recently I have problem with PCR amplification.
First i used High fidelity PCR master (Roche) to amply gene from genomic DNA, but failed when i tried to change different parameters (annealing temperature, different pirmers etc). When i used Taq enzyme do again, it works, i can see very nice band on gel.
Because of considering relatively high mutation of Taq, i tried to use pfu polymerase again (followed by mannual), but didn't work either.
So i wanner to know what's main points which differs results so much.


Biocat

Use a new batch of enzyme, or increase the amount of enzyme you put in.

More enzyme won't help if the conditions you are using aren't suited to the enzyme.

You will need to optimise the reaction. Try a range of annealing temps with a gradient run or a touchdown PCR, try a range of Mg concentrations, try a different extension time.

for interests sake, what conditions have you been using for both enzymes?

[ProteinWork]

swanny, on Apr 19 2010, 10:55 PM, said:

ProteinWork, on Apr 20 2010, 01:06 AM, said:

Biocat, on Apr 19 2010, 10:42 AM, said:

Hi all,

Recently I have problem with PCR amplification.
First i used High fidelity PCR master (Roche) to amply gene from genomic DNA, but failed when i tried to change different parameters (annealing temperature, different pirmers etc). When i used Taq enzyme do again, it works, i can see very nice band on gel.
Because of considering relatively high mutation of Taq, i tried to use pfu polymerase again (followed by mannual), but didn't work either.
So i wanner to know what's main points which differs results so much.


Biocat

Use a new batch of enzyme, or increase the amount of enzyme you put in.

More enzyme won't help if the conditions you are using aren't suited to the enzyme.

You will need to optimise the reaction. Try a range of annealing temps with a gradient run or a touchdown PCR, try a range of Mg concentrations, try a different extension time.

for interests sake, what conditions have you been using for both enzymes?

Yes, you are right.

I was just thinking that it might be that the pfu enzyme Biocat used is dead or denatured somehow. Because I use both Taq and pfu in my lab and I don't usually see such a distinct difference between the performance of those two.

To Biocat, have you tried the pfu to amplify other sequences to make sure the enzyme works? Look at the instruction manual of the enzyme and find out what the manufacturer suggests for positive control. If the enzyme is working, then try gradient or touchdown PCR as swanny said.

[Biocat]

ProteinWork, on Apr 20 2010, 06:17 AM, said:

swanny, on Apr 19 2010, 10:55 PM, said:

ProteinWork, on Apr 20 2010, 01:06 AM, said:

Biocat, on Apr 19 2010, 10:42 AM, said:

Hi all,

Recently I have problem with PCR amplification.
First i used High fidelity PCR master (Roche) to amply gene from genomic DNA, but failed when i tried to change different parameters (annealing temperature, different pirmers etc). When i used Taq enzyme do again, it works, i can see very nice band on gel.
Because of considering relatively high mutation of Taq, i tried to use pfu polymerase again (followed by mannual), but didn't work either.
So i wanner to know what's main points which differs results so much.


Biocat

Use a new batch of enzyme, or increase the amount of enzyme you put in.

More enzyme won't help if the conditions you are using aren't suited to the enzyme.

You will need to optimise the reaction. Try a range of annealing temps with a gradient run or a touchdown PCR, try a range of Mg concentrations, try a different extension time.

for interests sake, what conditions have you been using for both enzymes?

Yes, you are right.

I was just thinking that it might be that the pfu enzyme Biocat used is dead or denatured somehow. Because I use both Taq and pfu in my lab and I don't usually see such a distinct difference between the performance of those two.

To Biocat, have you tried the pfu to amplify other sequences to make sure the enzyme works? Look at the instruction manual of the enzyme and find out what the manufacturer suggests for positive control. If the enzyme is working, then try gradient or touchdown PCR as swanny said.

Thank you swanny and ProteinWork.
The proof reading enzyme I used should be working becuase my collegues also used these batches of enzymes and it works for their PCR amplification.
The problem for me probably is the reaction itself. I also amplied another gene with both Taq and High fidelity enzyme before, it looked fine.
But as far as the gene i used recently was concerned, I already tried different parameters:
Use different annealing temperature: 40, 42 and 50, 55C; different elongation tem: 68, 72; and also use diifferent elongation time as well.

[Vini]

well, if changing the conditions ain't helping, won't it be a nice idea to just get the PCR product from Taq sequenced to see if there are any mutations............if there aren't, then why bother with a different enzyme??

Also, you can maybe try increasing the numer of cycles....