Low Quality Seqeunces - (Nov/16/2009 )
Just wondered if anyone could recommend some reading/ background on why PCR's result in low quality sequences and things that you can do tot clean them up or steps required to get a high quality sequence?
For good sequencing you need a single sequence following the primer. If you have a good strong single band in a PCR reaction, then one of the two PCR primers can be used for sequencing (after purifying the DNA from the PCR reaction). If you have multiple bands or primer-dimers, you may need to gel purify the band of interest. The other major potential problem is the amount of DNA. Either too much or too little can cause bad sequencing results.
I had trouble with this batch of extractions I used a new kit and it gave me extremely variable results in terms of DNA concentrations so I had to dilute most of my samples to get the PCR's to work. I just looked back at my gels and I don't have much in the way of primer/dimer bands so it must be a problem of DNA quality, either too little or just dirty??
can you check the DNA concentrations of your PCR's like an extraction ?? would this help improve my sequencing results because then I wouldn't send crappy PCR's to be sequenced?