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Looking for Pfu Taq for high GC-content template


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#1 i_George

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Posted 22 February 2011 - 10:46 AM

Hi there,
I'm a new in molecular lab and new member in this board too, so pls help me..
I'm dealing with PCR cloning of templates with high GC-content (70.9, 67.3, 63.9, and 52.3% respectively)
I want my PCR product to be blunt-ended and proof-read
So far, I used Pfx50 DNA pol,invitrogen but it did not work at all

Some people talk about adding some additives, but I don't know where to start?
or do you guys have some Taq to recommend?

I'm looking forward to your responses

#2 phage434

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Posted 22 February 2011 - 01:58 PM

I would start by adding 5% of a 1 M Betaine solution.

#3 Adrian K

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Posted 22 February 2011 - 05:03 PM

My recent experience:
I was trying to amplify 2 highly GC rich genes for cloning, tried DMSO not working. Ordered betaine solution but not yet arrive. So I had asked for a sample of KOD extreme hot-start polymerase (with proof reading). Although there are many unspecific bands and smearing, but at least I still able to gel excise my desired band. Now it turns out to be the sequencing machine problem which can't sequence one of it.
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

"It's all just DNA. Do it."---phage434

#4 i_George

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Posted 22 February 2011 - 10:39 PM

I would start by adding 5% of a 1 M Betaine solution.


phage434--thanks for your reply
I will follow your advice
However,would you tell me what kind of betaine you use?
You mean Glycine bataine/trimethylglycerine, right?

#5 i_George

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Posted 22 February 2011 - 10:54 PM

My recent experience:
I was trying to amplify 2 highly GC rich genes for cloning, tried DMSO not working. Ordered betaine solution but not yet arrive. So I had asked for a sample of KOD extreme hot-start polymerase (with proof reading). Although there are many unspecific bands and smearing, but at least I still able to gel excise my desired band. Now it turns out to be the sequencing machine problem which can't sequence one of it.


Have you ever tried additive CES ?
I don't know if it works?

Edited by i_George, 22 February 2011 - 10:55 PM.


#6 Adrian K

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Posted 23 February 2011 - 03:33 AM

If you mean CES from:
http://www.protocol-...lates-3469.html
I would say I haven't tried it. I just tried DMSO since my betaine have yet to arrive.

The product I use: 71975 KOD Xtreme™ Hot Start DNA Polymerase
http://www.merck-che...74AAAEj2Bl9.zLX
It does have it's own propriety ingredients which make it work.

Here's betaine solution from sigma-aldrich (which I bought): B0300-1VL
http://www.sigmaaldr...RAND_KEY&F=SPEC
Have not tried it yet... my work was done so no worries.
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

"It's all just DNA. Do it."---phage434

#7 i_George

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Posted 23 February 2011 - 05:21 AM

If you mean CES from:
http://www.protocol-...lates-3469.html
I would say I haven't tried it. I just tried DMSO since my betaine have yet to arrive.

The product I use: 71975 KOD Xtreme™ Hot Start DNA Polymerase
http://www.merck-che...74AAAEj2Bl9.zLX
It does have it's own propriety ingredients which make it work.

Here's betaine solution from sigma-aldrich (which I bought): B0300-1VL
http://www.sigmaaldr...RAND_KEY&F=SPEC
Have not tried it yet... my work was done so no worries.


Here, in my lab we don't have betaine either.
I'm thinking that I should order betaine or a new DNA pol that can work well.
Which way is better you think?


Could you give me some reasons for using KOD Xtreme Hot Start DNA Pol. in stead of any other commercial ones?
So that I can tell my advisor about it efficiency and use it too
:)

#8 Adrian K

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Posted 23 February 2011 - 08:20 AM

I never tried other commercial proof-reading taq specially for high GC, so I can't comment much. I heard Roche have some products as such as well.

Pros for KOD xtreme: no need much optimization (cos you can't do much)
Cons for KOD xtreme: you can't adjust much (not flexible), expansive, no guarantee it will surely work.

Try betaine first, or if possible, ask Merck/other company to give you a sample to try...
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

"It's all just DNA. Do it."---phage434




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