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No amplification using pfu polymerase

PCR - Polymerase Chain Reaction Forum

PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.



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  #1 (permalink)  
Old 04-01-2007, 09:18 AM
Pipette Filler
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Question No amplification using pfu polymerase
Urgent help needed....
I am trying to amplify a gene from strep chormosomal DNA extraction to clone. Works beautifully with TAQ but when i try the same conditions using pfu hot start doesn't work at all.............Have tried manufacturers instructions..doesn't work!! getting desperate...Please trouble shoot...

Strep girl
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  #2 (permalink)  
Old 04-25-2007, 05:39 PM
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Default Re: No amplification using pfu polymerase
Have you noticed that pfu polymerase has lower extension rate?
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Old 05-24-2007, 10:10 AM
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Default Re: No amplification using pfu polymerase
I find it can help to try a range of MgCl2 concentrations in your PCR mix. You'd be suprised what an impact it can have.
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Old 05-28-2007, 09:48 AM
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Default Re: No amplification using pfu polymerase
It require very high qulity of your template DNA. Did you check your DNA qulity?
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Old 06-11-2007, 04:01 PM
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Default Re: No amplification using pfu polymerase
I had the same problem with pfu but by using a fresh DNA it worked very well.
try to purify ur DNA sample or fresh extract the dna.
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Old 07-10-2007, 11:27 AM
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Default Re: No amplification using pfu polymerase
i am really sorry to disturb you but i would like to tell you that right now i am working on an amplification of a gene with pfu and unfortunately it does not work so i was wondering what have you done and if you have resolved the problem. please let me know as soon as possible because these experiments are really important for my university thesis
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Old 07-16-2007, 02:01 AM
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Default Re: No amplification using pfu polymerase
hello

you should fresh DNA sample.IT works very well when fresh DNA sample is used.
You should also use a blend of Taq nad Pfu.I use 1:2 ratio of taq and pfu.
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