Hi guys!
I'm having to do a LOT of DNA sequencing and it appears the fastest way for me to do that is to set up 96 sequencing reactions and run them through the thermocycler, then taking them out and using isopropanol to precipitate (for 15mins-24hours) and centrifuging in batches of 24 before air drying them and sending them up to our sequencing facility.
Just wondering whether the function of the terminator premix will be affected if it takes more than a couple of hours to set up the 96 tubes.
(If anyone has some ideas to make the process more efficient it would be welcomed)
Thanks!
David
Hi David,
I also work with 96-well plates to do PCR and sequencing. I purify them by filtering in a PALL multi-well microfilter plate. You just have to filter your PCR product by pouring it into the plate well and then performing 10 minutes of vacuum or centrifuging. Once all the liquid is filtered, you ellute your PCR fragment in destillate water. It takes around 20 minutes and it's easy and reproducible.
I hope have helped you.
bàrbara.
Hi David,
The function of the dye-terminator premix should not be greatly reduced if it is left at room temperature for a couple of hours. The activity of the fluorescent dyes will likely not be reduced much if left in light for a couple of hours. Some factors to consider when working with any Taq polymerase includes the age of the premix and the number of times it has been freeze-thawed. Taq polymerase and big dye terminators are fairly stable over shorter periods of time. However, like any enzyme, constant freeze thawing and time could eventually reduce there activity. If necessary, you could simply aliquot into volumes.
We have very few multi-channel pipettes which are mostly used by the diagnostic department (we have had many staff cuts and are running behind - diagnostic work has a higher priority than research) and I'm using tubes not a plate. Making the reaction mix doesn't take all that long, but labelling all the tubes, then adding the template DNA for 76 samples can take a while. I'm not trying to say that it definitely will take me several hours, I'm trying to compensate for other things things such as interruptions which may occur during the process.
Ok, so as long as I avoid too many freeze thaw cycles I should be fine. Thanks for your help. :)
If there is any way that you can transition to using 96 well plates and a multichannel and or repeat pipettor, it will save you SO much time. Even just using 96 well plates so that you don't have to label individual tubes will help.
In my experience BigDye is pretty tough so I wouldn't worry about having it out at RT for a couple hours. Another thing - for sequencing reaction clean-up, I find that if the samples are left to precipitate for more than ~10 min at RT before centrifugation, I get dye blobs showing up in the sequence electropherograms. Sometimes this is an issue, sometimes not.
Good luck and I feel your pain!
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- DNA