Hello

Richard James r.james at uea.ac.uk
Mon Nov 7 08:42:54 EST 1994


> Hello Again ALF Users,

I hope to get a discussion going about ALF sequencers.

***Template Preparation***

What do users think is the best method for dsDNA template preparation
for the ALF?

We used to use Qiagen kits but they are quite expensive when used for
a lot of samples but give very good quality DNA when used with an extra ethanol precipitation step.

We have started to use the Pharmacia Easy Prep system, and after some initial problems (the vortex shaker for the plates is essential to get good yields), we are now happy with the system; especially if you buy the columns in bulk and make up the reagents.

***Fluorescent oligos***

We have recently purchased a Millipore Expedite DNA Synthesiser and are very impressed with the quality and the low price of the oligos. We now use the Millipore FluoreDite reagent for labelleing the oligos with fluorescent group detectable in the ALF. You can label up to 20 oligos from 0.1gm of reagent (which costs as little as $7 for each). We routinely make the oligos first, leaving on the trityl group, and store them in the fridge until we have made enough to set up a bottle of FluoreDite on the synthe
siser. We then add the fluorescent group to each oligo until the whole batch is done.

After purification by reverse phase these oligos give very good sequence data; as well as making primer walking much more economical.

***Sequence analysis***

We use the Lasergene software from DNASTAR for our seqeunce analysis on a Mac computer. The software is expensive but is very robust and can be installed on a local network to reduce the cost per user.

Regards,

Richard James
 
e-mail r.james at uea.ac.uk 
 




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