7 replies to this topic

[sara.r]

Hi

I need to amplify a 1.8 kb protein coding  gene for cloning and then expree it in mammalian cells, I used both pfu and long pcr mix enzymes (fermentas). pfu amplification was failed but I had sharp bands with long pcr mix, but I am still uncertain of feidelity of that mix. supplier says its error rate is 3 time less than tag, and it means that there may be some errors, I dont know what to do should I do more try with pfu or cloning 1.8 fragment amplified with long mix is safe?


regards

[Vini]

sara.r, on Jan 18 2010, 09:37 PM, said:

Hi

I need to amplify a 1.8 kb protein coding  gene for cloning and then expree it in mammalian cells, I used both pfu and long pcr mix enzymes (fermentas). pfu amplification was failed but I had sharp bands with long pcr mix, but I am still uncertain of feidelity of that mix. supplier says its error rate is 3 time less than tag, and it means that there may be some errors, I dont know what to do should I do more try with pfu or cloning 1.8 fragment amplified with long mix is safe?


regards

pfu might not have given u the amplification coz u did not use proper conditions....i think u can just go ahead with the long pcr mix enzyme.........if u r doubtful about the efficiency of the long mix, u can get the amplicon sequenced.......

[swanny]

Check the long PCR mix data sheet. You will find it's actually a cocktail of two enzymes, one of which (unnamed) is a proofreading enzyme. This is a standard product: the Taq gives speed, the proofreader gives fidelity. Typically, it's done in a 10: 1 Taq: proofreader ratio, which means that you can make it yourself for a lot less...

[leelee]

I would try and optimise the pfu reaction, better that you spend the time at this point rather than completing your cloning only to find an error!!
What conditions did you use?

I've been able to amplify 4kb using Finnzymes phusion which is high fidelity (in fact, it has a lower error rate than pfu, I haven't been able to find a better hifi polymerase out there!!)

Edited by leelee, 18 January 2010 - 11:30 PM.

[bob1]

leelee, on Jan 19 2010, 12:30 AM, said:

I've been able to amplify 4kb using Finnzymes phusion which is high fidelity (in fact, it has a lower error rate than pfu, I haven't been able to find a better hifi polymerase out there!!)

I use KOD polymerase - get more product with slightly better fidelity (according to the product sheet) than I do with phusion.

[leelee]

Haven't heard of that before bob, I'll check it out- thanks

[leelee]

I couldn't see anything on the product info about the error rate? Does it tell you on the sheet you have?
Thanks.

[bob1]

leelee, on Jan 19 2010, 06:56 PM, said:

I couldn't see anything on the product info about the error rate? Does it tell you on the sheet you have?
Thanks.

See attached sheet.  Though I notice it doesn't actually compare it to phusion, only to pfu (my memory playing tricks on me again... must be getting old).

Attached Files

•  KOD_polmerase.pdf   235.7K   295 downloads