I will be using the Himedia PCR cleanup protocol and kit, so if anyone has any hacks to reduce the smear, do let me know. I will be sending samples urgently for sequencing. The sequencing is chance-based; sometimes the results come, and sometimes they don't. Even if the DNA purity and band smears are the same, please help.
I would say if you are really interested in the sequence then perhaps lower target input or doing a nested PCR where the first PCR has primers then that is diluted and used in a second PCR with primers internal to the first. might get more specific binding. or perhaps increasing the annealing temperature/ lowering salt to reduce non-specific binding.
you can increase 2 or 3 degree Annealing temperature for PCR. it will help to remove unwanted bands and smears. I often try this hack. Good Luck!
You have primer dimer so using less primer or a hot start polymerase will help. The bands look over amplified so fewer cycles or a hotter annealing temp or using less dna template will help. The smear will purify with the pcr band so purification of the band cut out from the agarose gel and column purified should give a clean template and good sequencing
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