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long range PCR


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2 replies to this topic

#1 sud373

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Posted 22 March 2010 - 11:45 PM

hi all,
i want to amplify 6 kb long DNA frgment. & for that I have designed 2 set of primers. for first set of primer suggested anneling temp is 54(FP)and 56 degree C and for the second set it is 60 and 61 respectively. I am unable to amplify the GENE. I also incresed the dNTPs concentraion but didnt get amplification.
PCR progrom i kept is ..
1. temp 95 C for 5 min
2. 94 C for 1 min
3. 54 C for 1 min
4. 72 C for 6 min
5. goto step for 30 times
6. final extension for 10 min
7. 4 C for ever

Please help me out.
suddu

#2 Vini

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Posted 23 March 2010 - 01:00 AM

hi all,
i want to amplify 6 kb long DNA frgment. & for that I have designed 2 set of primers. for first set of primer suggested anneling temp is 54(FP)and 56 degree C and for the second set it is 60 and 61 respectively. I am unable to amplify the GENE. I also incresed the dNTPs concentraion but didnt get amplification.
PCR progrom i kept is ..
1. temp 95 C for 5 min
2. 94 C for 1 min
3. 54 C for 1 min
4. 72 C for 6 min
5. goto step for 30 times
6. final extension for 10 min
7. 4 C for ever

Please help me out.



which polymerase are you using???? have you tried Roche's, Expand long template PCR polym.????

#3 phage434

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Posted 23 March 2010 - 04:37 AM

I second the recommendation. What is the template DNA?




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