I recently posted the message below to the group. All of the responses
were sent to me directly so I though I would post a follow up so that
anybody interested could see what people had to say. Thanks to everyone
who responded.
Shaun.
We have recently switched over to the Big-Dye chemistry and I have to
admit that I=92m pretty impressed. However I=92ve recently come across
something which leads me to believe that the new chemistry may not be
quite as robust as the old one. I=92ve been having some problems with
sequencing some PCR products cloned into pCRII. The amplicons are
approximately 700 - 800 bp and the plasmids are being purified using
Promega=92s Wizard Plus system. I=92ve sequenced a number of similar
clo=
nes
in the past with the old chemistry using exactly the same procedure and
the results were great. But now I=92m getting either very weak signal or
no signal at all. The plasmid preps are quantitated by A260 and I=92m
using the same amount as before. I=92ve had some moderate success in
improving results by using the cycling conditions for cosmids (i.e.
increasing the cycles to 60) recommended by Bruce Roe (I=92ve been using
this for sequencing some rather large plasmids and stumbled on this by
accident -- you can probably guess how). I=92ve also had some luck by
simply reprecipitating the plasmids with EtOH after extraction. I
suspect that there may be something in the purified plasmid obtained
with the Wizard kit which is partially inhibiting the sequence reaction
but this was never a problem with the old FS chemistry.
Any comments or suggestions you may have would be appreciated.
Shaun Tyler
DNA Core Facility
Laboratory Centre for Disease Control
Health Canada
Ph#: (613) 941-6441
FAX#: (613) 957-1358
E-mail: styler at hpb.hwc.ca
> Hi Shaun,
>> We've had a few users here in Australia that have seen the same thing. The
> problem has proved to be with the Wizard kits - they don't seem to like the
> BigDye Terms. Once these users changed to alternate purification methods, the
> BigDyes almost blew the front off their monitors.
>> Regards,
>> Dan Headon
> PE Applied Biosystems Australia
>>>> Hi Shaun,
> Glad to see someone visits my web page and has something nice
> to say.. :-)
> A couple of things to suggest, look at the protocol we are
> using for the ds template isolations. Essentially it's a quick and
> dirty (actually rather clean) cleared lysate *without* all the extra
> stuff like Wizzard or filtering or phenol.
> Second is to do a template concentration study with the Big dye
> reactions. Quite supprisingly, we and others have observed that 2 to 5
> times *less* template than was used previously with the dRhodamines seems
> to give much better results in most instances. Strange but rather true.
> Third, for regions that are very GC rich, it seems that going
> back to the dRhodamines often reads through them when the Big Dyes
> fail. Oh BTY, we always use either 5% or 10% DMSO in both the dRhodamine
> and BigDye reactions and are use the dRhodamines at 1/4 reaction (5-7 ul
> total reaction volume) and the Big Dyes at 1/16 reaction (also a 5-7 ul
> total reaction volume).
> Last but not least, it seems very clear from lots of expts. that
> EDTA inhibits the Taq Polymerase. Thus, instead of dissolving our template
> DNA in TE or diluted TE, we now routinely are just using sterile ddH2O.
> I'd much rather keep the EDTA there to inhibit nucleases but its inhibitory
> effect is severe enough that steril ddH2O is a better choice.
> Hope this helps and you might want to pass this on to the group
> if you think it useful.
>> Cheers........bruce
> *************************************************************************
> Bruce A. Roe, Ph.D George Lynn Cross Research Professor
> Department of Chemistry and Biochemistry
> University of Oklahoma, Norman, OK 73019-0370, U.S.A.
> Phone: (405) 325-4912 or 7610; FAX: (405) 325-7762; e-mail: broe at ou.edu> ********************** http://www.genome.ou.edu/ ************************
>>>> Hi Shaun,
> I direct a Core Facility - that sequences templates brought to us by
> many different users every day.
> We too seem to have had a problem with a higher number of "failures" -
> i.e.
> no real signal or too low to read signal with templates that appear to
> have
> plenty of DNA leading me to the thought of potential trace inhibitors in
> the template.
> This seems to have occurred after our shift to "Big Dye" a few months
> ago.
> It has been hard to trace because of so many different
> preparation methods - could you let me know your thoughts on any
> responses you get?
>> I have been telling people to beware traces of ethanol and/or EDTA - but
> I
> don't really know if that is the explanation.
>> Sorry I don't know more.
> Susan
>> Susan Hollingshead, Ph. D.
> Department of Microbiology, BBRB 654
> University of Alabama at Birmingham
> Birmingham, Alabama 35294
> Phone (205) 934-0570
> Fax (205) 975-5480
> Email: hollings at uab.edu>>>> Shaun,
> I was interested to read your e-mail to the newsgroup. We used to use the old primer chemistry with the Promega DNA isolation in our high throughput lab. I spent 3 months trying to implement Big Dye changing just about every component with no success until we changed to Qiagen REAL. It started working great! So we've stopped using Promega but we are now, on what seems to be a fairly regular 10-14 day cycle, getting some similar problems of very low signal intensity, it is also associated with the quality of the minipreps as viewed on agarose gels going down. What do I mean by going down? Well the quantity may go down, or we may get what looks like a scrambled DNA effect or even nothing. The strange thing is even if the prep looks good on the gel the intensity on the 377 gets lower over time. This has happened to us twice now and we've redeemed the situation by throwing everything away and starting again( initially we thought we had a contamination, but the micology came!
b!
ack negative), the preps look good and consequently the sequence. Unfortunately I haven't got the luxury to test each component one at a time so I haven't identified the culprit yet. So I'm beginning to wonder if it's not the DNA extraction but the "age of the plating cells or another factor associated with the XLOLR's, and in fact the Promega might be ok. I took over running the facility here at Genesis about 7 months ago and some of the procedures have some unclear history behind them, one is that they've always put maltose (autoclaved not filter sterilized) along with Mg2SO4 in NZY broth to grow the XLOLR's. Now we're infecting the XLOLR's with an M13 based virus, so no maltose receptor required and no Mg to stabilize the heads? Anyhow I'm growing up the bugs today without either so we'll see how that goes. If you have any other suggestions I'd be clad to chat about them. So my initial advice is try a different miniprep kit, the REALs weren't as good as the Turbos but the!
p!
rice is a lot cheaper. Tell me how you get on
> Cheers Matt
>>
