4 replies to this topic

[Juliasarmoire]

My master's thesis is killing the biologist in me I'm in the point of wondering could I possible write a thesis how NOT to isolate this protein, so things are bad... everything looks perfect, but the sequencing results are awful, my 1kb band is full of random mistakes and gaps and I suppose the only logical step is that my PCR isn't working properly. I got 500bp band nicely out (with Taq)... but with 1kb band, the error started. I used Taq polymerase and switched to phusion enzyme from Finnenzyme which just made things more interesting...

I use human cDNA library as a template (100ng)
I have changed the buffer, which didn't change anything.

I use 2 min 30 sec initial denaturation time 98 C as I suppose human cDNA counts as a "complex" template
denaturation is 30 sec at 98 C
annealing 20 sec at 63 C
extension 40 sec at 72 C
and final extension 10 min at 72 C

I have exactly that one 1 kb band and everything looks perfect... then I do gel extraction, digestion, purifying, ligation, transformation, plasmid purifying, digestion to see if I have the right insert... and my 1kb bands are looking fabulous... then I sent them for sequencing and receive back some nonesense. I switched from Taq to phusion because Taq made a few mistakes... but this phusion... it's not doing it's job or maybe I really can't use it right Others get fabulous results with it and me... has anyone ever worked with human cDNA libary and a phusion enzyme? Is there some kind of special tips how to make this working... in one of the samples the phusion had already a gap in the third nucleotide in the primer... so it would seem that the annealing time is too short? But then again... the gaps are random. Sometimes in the middle, sometimes in the end and sometimes in the beginning... I have double checked the reactions to make sure that I didn't have any contaminations... I have tried everything... maybe there is something wrong with the cDNA libary....

Edited by Juliasarmoire, 29 October 2009 - 06:40 AM.

[fishdoc]

I had a similar problem within the past couple months with Phusion (see thread here).

I ended up using another high fidelity polymerase, and had similar but less mutations. I changed out my DNA template, and things worked better after that. Since then have used Phusion with no problems that I know of, but I am not sequencing as much as I was, either.


I didn't have trouble with gaps, but with incorrect bases. Have you checked the chromatographs to see if the sequencer just missed the calls?

Edited by fishdoc, 29 October 2009 - 07:14 AM.

[ivanbio]

Hi Juliasarmoire,

Don't despair, research is all about struggle. Likely hearing this does not help at this point, but trust me, all of us that spent any time at the lab have gone through experiments that simply did not want to work.

You seem to be performing all the right experiments/tests so I am going to offer you an alternative. Since you point out that you have a 1 kb insert and you are able to identify errors all over the place, I assume you know the exact DNA sequence you are looking for. If that is the case, I would recommend you send that sequence to a company that can chemically synthesize the whole 1 kb insert for you. These days that will cost you about $400-$500. I've used this approach very successfully in the past.

Plus, if you think about it, there are so many other experiments you will want to do afterwards with your purified protein that spending too much time on this step is simply not worth it.

Good luck!

Ivan

[phage434]

It sounds to me as if your sequencing is failing, but not your cloning.  Are you sequencing with a primer spaced away from your cloning site by 50 bp or so?  Are you relying on the AGCT reads in text form, or are you looking at the electropherogram?  Are you sequencing from both ends?  How are you checking the DNA concentration you are sending for sequencing?

[Vini]

Juliasarmoire, on Oct 29 2009, 08:08 PM, said:

My master's thesis is killing the biologist in me I'm in the point of wondering could I possible write a thesis how NOT to isolate this protein, so things are bad... everything looks perfect, but the sequencing results are awful, my 1kb band is full of random mistakes and gaps and I suppose the only logical step is that my PCR isn't working properly. I got 500bp band nicely out (with Taq)... but with 1kb band, the error started. I used Taq polymerase and switched to phusion enzyme from Finnenzyme which just made things more interesting...

I use human cDNA library as a template (100ng)
I have changed the buffer, which didn't change anything.

I use 2 min 30 sec initial denaturation time 98 C as I suppose human cDNA counts as a "complex" template
denaturation is 30 sec at 98 C
annealing 20 sec at 63 C
extension 40 sec at 72 C
and final extension 10 min at 72 C

I have exactly that one 1 kb band and everything looks perfect... then I do gel extraction, digestion, purifying, ligation, transformation, plasmid purifying, digestion to see if I have the right insert... and my 1kb bands are looking fabulous... then I sent them for sequencing and receive back some nonesense. I switched from Taq to phusion because Taq made a few mistakes... but this phusion... it's not doing it's job or maybe I really can't use it right Others get fabulous results with it and me... has anyone ever worked with human cDNA libary and a phusion enzyme? Is there some kind of special tips how to make this working... in one of the samples the phusion had already a gap in the third nucleotide in the primer... so it would seem that the annealing time is too short? But then again... the gaps are random. Sometimes in the middle, sometimes in the end and sometimes in the beginning... I have double checked the reactions to make sure that I didn't have any contaminations... I have tried everything... maybe there is something wrong with the cDNA libary....

hey, i don't think anything is wrong wid d enzyme. phusion works really well, it is high-fidelity enzyme and gives excellent performance. are u sure that u r following the instructions given in its pamphlet properly? as phage434 suggested, it seems more to be a problem of sequencing....