Automated sequencing problems (ABI)

Todd Martinsky techmen at technologymentors.com
Tue Dec 16 11:47:31 EST 1997


Dear Harry:
This protocol came from our friends at deCode Genetics.  They are confident
it is the best method out there, because they have tried them all.
Hopefully it will work for you too.
Good luck in finding all your answers.
Regards,
Todd

Protocol for purifying PCR products with the
ArrayIt PCR Purification Kit:   Specifically for sequencing with Perkin
Elmer 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.
This protocol has been developed by our colleagues at deCode Genetics.  We
continue to be confident that if you try other methods including kits from
other manufacturers, TeleChem's will work the best and save you money. As
you can see the main difference in this protocol is that we are using a
centrifuge instead of vacuum.
TeleChem can be contacted at:
TeleChem International, Inc.
12 S First Street, Suite 817
San Jose, CA 95113-2405
Telephone 408-977-0160 Fax 408-977-0164
Email technical questions to: telechem at hooked.net
WWW:  http://www.hooked.net/~telechem/arrayit/
Harry Witchel wrote in message <34967E6F.88F9E28D at Bristol.ac.Uk>...
>Hi folks!
>
>I currently use an in-house facility for sequencing (ABI 377, cycle
>sequencing), which is quite reasonably priced.  However, it is extremely
>common for them to be unable to sequence my templates.  This is often
>true for standard primers (eg M13 uni and reverse) as well as other
>primers.  My templates are often quite GC rich (65%).  I purify my
>plasmids using Promega’s Wizard Plus kits, which have (at times) given
>me very good sequence data with the same sequencing facility.  I always
>use the same strain of bacteria (XL1 MRF’), although every few months I
>have to make a new batch of competent bacteria.
>
>The inconsistency of my results disturbs me, and I want to increase my
>quality control.  I always check my plasmids before sending them out for
>sequencing (thus checking for plasmid concentration & for the ability to
>be restriction cut).  I am now trying to keep track of all the variables
>that might affect sequencing, which so far include: Date, Miniprep type
>and batch #, Sequencer, Primer, Bugs, Medium, Vector.  If anyone knows
>of any other important variables, I would appreciate it.
>
>Finally, as I am trying to do some quality control and cost savings, I
>am trying to figure out what is worth doing.  I have used Qiagen
>Tip-20s, and they cost 6x as much as the Wizard preps, and at times they
>did not work (but I never did a formal analysis of why or what percent
>failed compared to Promega).  Any help managing this problem and
>increasing my sequencing success is appreciated.
> Thanks,
>  Harry
>
>
>
>--
>Harry J. Witchel, Ph.D.
>Dept. Physiology
>Medical School
>Bristol  BS8 1TD
>   England
>
>Harry.Witchel at Bristol.ac.uk
>http://www.bris.ac.uk/Depts/Physiology/Staff/hw.htm
>
>





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