PCR High GC content - PCR High GC content (Nov/16/2005 )

hi
I need to amplify High GC content targets (65-68%). If I do hot start and I get results but it is cumbersome to do with large samples. please give me some suggetions to amplify these targets ( preferealby not using vary expensive kits)
seeking help
shashi

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I'm currently working on myccobacterial DNA which is very high GC content and I use the normal T aq from Quiagen together with their Q solution (remove some weird secondary structure) and it works fine

Pesji

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Hi
can u tell me the PCR mix you ar using --I mean componenets, do u use DMSO

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[quote post='31404' date='Nov 16 2005, 08:45 AM' name='pesji']


I'm currently working on myccobacterial DNA which is very high GC content and I use the normal T aq from Quiagen together with their Q solution (remove some weird secondary structure) and it works fine

Pesji
[/quote]

Hi
can u tell me the PCR mix you ar using --I mean componenets, do u use DMSO
[/quote]
I'm using all reagents given with the Taq enzyme from Quiagen as far as I know it doesn't contain DMSO but the Q solution is probably playing the same role !

Pesji

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Hi,

Did u have problen in Annealing?

Here a formula for Annealing temp.

Temp of Annealing = +/- 5 C temp of Melting

Temp of Melting = 3 ( G+C ) + 2 ( A + T )

And use Q solutions for ur PCR form Qiagen. It 'll adjust the temp of annealing and give good result.

Best of luck,

Bye ,

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For difficult templates, I often use an additive as well -- the PCRx Enhancer System from Invitrogen. It works well (review here). Instead of a hot-start PCR, I'll use one of Invitrogen's Platinum Taq's or Platinum Supermixes -- they include an antibody that holds the Taq inactive until the antibody is denatured by the first high temperature step of your PCR protocol, thus allowing you to prepare your reactions at room temperature.

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5-10% DMSO w/ 10% Glycerol works for me...good luck

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I add 10% (v/v) of DMSO 50%(w/v)...and the signal is perfect!!My template has more than 65%of CG!!

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I believe the wonder agent in Q solution and other soloutions like it is Betaine. You can buy this seperately and add it to your PCR.

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Hi Shashi
for amplifying DNA containing high GC content, you must use DMSO or formamide (2% to 10%). For more discussions you can contact me at tryprabhanshu@tahoo.co.in or at 09868613020 in INDIA
bye
Prabhanshu Tripathi

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