Dear Andrew,
I work in a sequencing facility at the Albert Einstein and we routinely
sequence and run stuff on 384 well plates.
This is how we precipitate stuff in our facility :
1. To a 10ul seq reaction add 25ul 100% EtOH at room temperature
2. Incubate at ROOM TEMP for 45 min.
3. Spin at 4000rpm for 45 minutes (we use plate carriers in an Eppendorf
centrifuge ).
4. To remove ethanol, turn plates upside down on paper towels and spin for
30 sec. at 200rpm.
5. Now add 15ul of 70% ethanol - RT - to each well with multi channel and
spin at 4000rpm for 10 minutes.
6. Remove ethanol as described.
7. Repeat 70% wash as described.
8. Then remove ethanol again as described.
Using the above procedure we get good recovery and read lengths in excess
of 500bp at 100% accuracy for good templates.
The exclusion of salt in the precipitation was evaluated and seems to have
no obvious effect on our recovery. However, bear in mind that our DNA for
sequencing is of a crude miniprep grade and therefore some salts may come
through from the samples themselves.
In addition, the exclusion of salts not only predisposes to longer reads by
leading to a more 'focused' run on of product and therefore single base
pair resolution later on but also the signal strength is more balanced by
virtue of being stronger at the 3'end. Remember, the electrokinetic
injection is very salt sensitive and salt retards transfer, particularly
the longer products.
The use of room temperature ppt. minimises ppt of salt and is therefore
good for the above reasons.
Hope this helps.
Laurence
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Laurence Hall,
Einstein Genome Centre,
1695 Poplar Street,
New York 10461.
Tel. (00) 1 718 405 8380
Fax. (00) 1 718 405 8383
E-Mail : LHall@megabase.aecom.yu.edu
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