Insta prep for sequencing
techmen at technologymentors.com
Fri Dec 5 14:19:33 EST 1997
I don't know if it is or not, but TeleChem http://www.hooked.net/~telechem/
has produced PCR purification system that is being used for Sequencing with
Dye Terminators, Perkin Elmer ABI-377. Protocol included here...
Protocol for purifying PCR products with the
ArrayIt? PCR Purification Kit:
Specifically for sequencing with Dye Terminators from Perkin Elmer on their
ABI-377 automatic sequencers.
1.Pipette 100 ul per well of ArrayIt Binding Buffer with a multichannel
pipette to a 96 well plate
2.Add 25 ul of your PCR product to the well and mix it by pipetting 10 x.
3.Pipette the 125 ul contents of each well to an ArrayIt Super Filter and
position it on the empty 96 well plate.
4.Spin at 500 g for 5 sec. (Here we use Sorvall Tabletop RT-7 with RTH250
rotor and 96-well tray adaptors).
5.Move the ArrayIt Super Filter to a deep well plate (Here we use titerblock
from USA Scientific).
6.Wash each well of the ArrayIt Super Filter immediately with 350 ul of
ArrayIt Wash Buffer. (Here we use a Distriman repeater pipette from GILSON).
Spin at 500 g for 5 sec. Repeat twice dispensing the wash buffer after each
spin. Spin the last spin for 6 min to get rid of all wash buffer.
7.Transfer the Super Filter to a marked 96 well plate.
8.Add 100 ul per well with the Gilson pipette of 0,1X TE (ph=8,0).
9.Let stand 1-2 min. to allow re-wetting of the membrane.
10.Spin at 500 g for 5 min.
11.Put the ca. 80 ul samples in 55-C to dry. This usually takes at 2- 3
hours or overnight. At this step a 96-well plate vacuum centrifuge would be
very useful but we don't have one yet.
12.Resuspend the DNA in 25 ul dH2O or buffer of choice.
Virginia Wotring, PhD wrote in message <348831FC.1853 at nrc.uab.edu>...
>Does the 5prime-3prime product Insta prep really produce DNA that can be
>used for sequencing? Their ads/catalog claim that this is true. Has
>anyone used this product for sequencing? Was DNA purity and quanitity
>both good enough to sequence?
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