Today I ran a high
sensitivity DNA bioanalyzer chip to quality check some amplicon
libraries I was setting up for the MiSeq. In so doing, I noticed that
my peaks were coming out at the wrong sizes, for a simple reason; the
2100 Expert software hadn't correctly detected one of the ladder
peaks (the second, 50bp band).
Note second peak from left has a peak concentration of zero! |
It hasn't been recorded as the 50bp ladder band, instead mistakenly recorded as 39bp |
I had a look through
the user
guide and the troubleshooting
guide (see page 41) to try to re-assign the ladder peaks as you
can set the upper and lower marker using manual integration, to no
avail. A quick google and check of SeqAnswers didn't help either.
In case anyone's
struggling with a similar problem/lack of immediately searchable
answer, here's how I solved it.
The problem was really
that the software had called the two biggest peaks at a location that
really was just the biggest; presumably it looks for the upper and
lower markers first, then assigns the remaining peaks from largest to
smallest.
This is easily solved -
I just right clicked the peak (on the Peak Table tab when looking at
the electropherogram) and exclude it. After that the program
re-calculates the proper sizes, assigning the 50bp peak correctly and
outputting the correct sizes for my samples. Job done!
The slight shoulder off the upper marker mistakenly got detected as the next peak down |
Much better! |
Now the band is correctly labelled |
This is probably an
obvious fix, but if (like me) you were looking for the wrong search
terms (re-assigning peaks when really I needed to exclude) hopefully
this might help you shave a little bit of time off for you!
No comments:
Post a Comment