Im trying to amplify a fragment that include ITRs of AAV, from plasmid.
But i have a lot of problems.... And i have problems trying to sequence ITR, we use Macreogen service, any suggestion?
Regards
Anamaria, ITR is notoriously difficult to PCR. Check this: http://www.biotechniques.com/BiotechniquesJournal/2014/May/Comparative-next-generation-sequencing-of-adeno-associated-virus-inverted-terminal-repeats/biotechniques-351745.html
If you can't use MiSeq, try to use PCR system for GC-reach sequence.
AAV ITRs are extremely difficult to PCR or sequence - I have even had a AAV expert tell me not to bother because it won't work (never say never I guess?)
It may be worth trying to get your target sequence synthesised if you have persistent problems? The company may not be able to sequence the product for you but it could be worth a try.
I would amplify the fragment as a plasmid in a Stbl2 line (designed to be stable despite repeat regions) and try to use other cloning techniques to introduce your mutations/inserts/whatever. Are there any convenient cut sites nearby?
As was said previously, sequencing fails in repeat sections.
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