long range PCR - (Mar/23/2010 )
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.
-sud373-
sud373 on Mar 23 2010, 02:15 PM said:
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.
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.????
-DRN-
I second the recommendation. What is the template DNA?
-phage434-