3700 384-Well Platform

Dr N.I.Leaves nleaves at hgmp.mrc.ac.uk
Fri Aug 24 04:33:45 EST 2001


On 23 Aug 2001, jws36065 wrote:
> Hi,
> Our Sequencing group is currently in the early stages of looking into
> converting our 3700s for a 384-well format.  The beginning steps will
> involve validating a few PE 9700 Thermal Cyclers for use in the cycling
> rxns, etc.  
> Any feedback on steps to take, protocols, etc would be greatly appreciated.
> Thanks!
> 
> ---
> 
> 

Hi,

we've just gone down this route and its been very successful. We use MJ
tetrads for cycling. 

In our lab we found that the sequencing clean up has been tricky in the
384 well format - ethananol/EDTA precipitation did not give us good
results, we thought because of a mixing problem in the small wells. We now
use a new 384 well product from Millipore for this which gives lovely
results. We buy our sequencing plates form ABgene and use them on the ABI
black plate bases supplied with the machine.

Our only other problem was handling/creating sample sheets for 384. We
make our DNA in 96 well plates and our robot combines these, interweaving
them on a 384 well plate. The 3700 then loads 1/4 of each 384 well plate
to the calliparies. This means each 3700 run gets what is effectively 1/4
of a 96 well plate. We wrote some software to reorder all this data
back into their original 4x96 well format when a complete 384 plate is
finished. Does that make sense?

Other interesting observations... 

You will be running longer unattended, and its very easy to underestimate
how much, buffer, water and POP you use. Each of our 384 well plates takes
us about 11 hours so we get more than 2/day. We pad out the machine with
occasional 96 well plates. We now regularly get 55 runs because we load 3
x 384 well plates on a friday night and someone comes in on Sunday to load
another 2 plates. We've tried loading 4*384 but ran out of water on the
4th plate. ABI say there is an upgrade... (a bigger water
container!?). Make sure you check under "plate setup" "preferences" that
your batch runs are permitted to go for longer. When we first started 384
plates we didnt realise under this preferences we were limited to 4 runs 
in 24 hours and the machines kept stopping... its easy to change.

Good luck

Nick

end 
************************************************** 
Dr N I Leaves
Mouse Sequencing
MRC HGMP Resource Centre 
Hinxton 
Cambridge CB10 1SB 
tel: 01223 494557 (office) or 01223 494541 (lab)
email: nleaves at hgmp.mrc.ac.uk
**************************************************




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