Long PCR Protocol
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Long PCR Protocol
The key to LA PCR is an enzyme, TaKaRa LA TaqTM. It is a thermostable DNA polymerase, which possesses 3กฏ to 5กฏ exonuclease activity, or proofreading activity. The efficiency drastically declines when incorrect bases are incorporated. The 3กฏ to 5กฏ exonuclease activity removes these misincorporated bases and makes the further reaction proceed smoothly. Therefore, the amplification of long DNA fragments can be achieved. (Alam lab, University of Hawaii at Manoa)
Last update 24-Aug-2006, Rating Very Good of 2 votes.
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Long PCR is for PCR amplification segments larger than 2KB in lenth. It's always difficult to amplify for some reasons such as :
1.Taq polymerase lack an editing function, mismatched base at 3' end of a extention strand.The new improvement polymerase (ne.g. pfu) can help to correct mismatched end.
2.the template is easy to be damaged during high temperature in buffer. this problem can be fixed by increasing Tris-HCl to 250 mM in a 5X PCR Buffer, PH is 9.0. other components include glycerol 5% in a final reaction 0.01% gelatin in a final can be added to help long PCR amplification
3.Use long er time to denature very long DNA molecular.
A sample for long PCR:
94C 3 min
30 cycles of
94C 1 min
60-67 C 1min
68C 5-20 min
68C 7 min
Rating: Very Good
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For Long PCR, It's better use touchdown PCR program.my protocolsfor 3-5kb cDNA amplification is ( use roche kit):
93-95 C 3-5 min ( up to different DNA polymerase)
10 cycle of
93-95C 20''
55C-65C 30''-45''
68C 5-6 min
20 cycle of
93-95C 20''
55C-65C 30''-45''
68C5-6 min Plus 20'' each next cycle
68C 7min
very good results.
If you could not get good PCR, tru to re desigh Primers
Rating: Very Good
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Related resource
Long PCR of circular mitochondria DNA
Long PCR Reagents and Guidelines
Troubleshooting for Long Template PCR
PCR fidelity of pfu DNA polymerase and other thermostable DNA polymerases
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