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In the Sequencing Core, we examine virtually every lane from every
sample we sequence
(excepting only some high-volume bulk jobs where we negotiated minimal
technician
involvement). We diagnose the problems whenever we can, and some
problems just keep
coming up again and again.
In addition, the Director spends considerable time with
clients who have had problems,
and carefully examines their experimental design and their protocols
for the cause
of the failure. The following table is a compilation of the most common
failure modes
seen in the DNA Sequencing Core.
Here are the
most common reasons that sequencing samples fail:
- Inadequate template concentration
This is SUCH a common problem, I'll break it down into
sub-categories:
- trying to spec a miniprep plasmid
- spec readings too low to be accurate
- inexperience at gel estimation of band intensity
- failure to check PCR product concentration
- spec reading with excessive RNA present
- spec reading with excessive genomic DNA present
- Overloaded miniprep purification devices ("Loss of
Resolution" samples)
- Failure to streak clones to single-colony
- Calculation error in primer concentration
- Multiple priming sites present
- Multiple inserts present (gives multiple priming or
stem-loop)
- Sequencing incorrect PCR products
- Failure to fully characterize a new plasmid construct
- Secondary structure in clone (siRNA constructs, etc)
- Homopolymer tract (e.g. 3' sequencing on a cDNA clone)
- Salt contamination in sample ("ski-slope" effect; typically
gel-eluted frags)$a
- Mis-paired primers (cross-species primer design, inaccurate
sequence basis)
Less common,
but still significant:
- Free dNTPs present in template
- Primer dimers
- Primer Tm too low
- Mixed-up samples and/or primers (i.e. mixed up in the client's hands).
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