The follow are just some comments on the recent postings related to the
3700. First I wanted to suggest that people might supply some or all of
the following information when relevant.
1)How many runs on the capillaries in total and since the last acid wash
and maybe an estimate on how frequently the capillaries were washed
2)Your conditions for loading your samples(buffer used, injection
parameters and foil piercing or not)
3)running conditions (polymer type and lot number, voltage, capillary temp
and cuvette temp).
4)an estimate on range of phred >20 scores if you use it.
Our 3700 is currently being checked out by ABI(PEB, perkin elmer etc.)
because of our concerns on how consistently the machine performs. I have
not run it for a while now and the above parameters were changed during our
trouble shooting period so I won't list these things at the moment.
On the observation by Stuart that the data quality tails off at 200-300
bases. I think the two possible conditions you describe is that there is
signal but the resolution becomes poor or that the signal is top
heavy(signal at start that fades too early).
On the top heavy signal you might try running the sample again. If
it works better the second time it would suggest that there were too many
small ions either because of the sequencing reaction (which you should see
on the gel also) or because of salts in the sample. Many people still
resuspend their templates in buffer which I believe does not help and EtOH
percipitation on the cleanup can leave salts in the sample. You should
also consider your loading solution and your injection parameters.
If it is a poor resolution it can be either the capillary or the
sample or both. We have found (after running ALOT of pGEM plates) that
after an acid wash we get a good consistent phred20 (between 650-700 on all
capillaries) but after about a week that the phred can range from 600 to
350 on pGEM and this appears to have a general left to right trend. The
lower phreds being on the right capillaries. We also developed a problem of
a totally random poor resolution on about 40% of the capillaries per run.
This appeared to be resolved by changing the board that controls the
loading robot and syringes???
My understanding is that the inline filter is only for the polymer
going to the cuvette for the sheath flow. The capillary fill is not
filtered so bubbles can be a problem. I think just being careful when
changing polymer and also be sure that the polymer fill syringe does not
leak when filling is the best you can do. If you get a big bubble in the
line I would run the change polymer wizard. I did see big bubbles develop
in the POP5 but I don't think they got into the line.
On reloading the sample, in the past this worked pretty good. I
have seen data where we ran the same plate three runs in a row and the
phred scores practically overlayed each other when we plotted them.
However more recently I found that the Gs were going off much quicker(I was
loading in water with no foil piercing on all runs).
We found that running a whole plate of pGEMs was very informative
on capillary to capillary performance and I would encourage everyone to
have some similar reference point. It is a pity the the Bigdye standards
are so unreasonably priced.
Hope my ramblings were of some interest. Looking forward to seeing
other comments. Thanks
Martin
This archive was generated by hypermail 2b29 : Thu May 18 2000 - 09:47:35 EDT