Loading dyes - thanks

Scottie Adams sadams at northnet.org
Mon Jul 17 10:24:08 EST 2000


Thanks to all the kind and generous people who took time out from their
busy days to answer my qustion about Loading dyes.  I have compiled the
answers below for anyone that is interested.  Names have been removed to
protect the innocent.  :-)

Scottie

Scottie,

Check out www.microzone.co.uk I know they sell a buffer with the pink dye
and are a lot cheaper than the 20 pounds per ml that Amersham wanted last
time I looked.

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Here are some sites to find loading dyes:
(Note: I am assuming that you are using 377s)

http://www.genome.ou.edu/
Look under protocols; look at 377 protocol:
http://www.genome.ou.edu/ABI_Prot3-9-98.html
4) Deionized Formamide
In a beaker, use 10g of Amberlite per 100 ml of formamide. Stir this solution
for 1 hour to deionize. Filter the solution using the 150 ml Nalgene
filtering system. Store in a dark bottle in the ABI refrigerator.
Note: if you already have deionized formamide, then all you have to do is add
formamide to get an 80% formamide:20% 25mM EDTA, 50mg/ml Blue Dextran
solution.  i.e., 4 mL of formamide, 1 mL EDTA/Blue Dextran.
5) 25mM EDTA, 50 mg/ml Blue Dextran
Add 0.93g of EDTA to 90 ml water. Then, adjust the pH to 8.0. Bring the final
volume to 100 ml. Next, add 50 mg Blue Dextran to a 1 ml EDTA solution.

Note: you can change these volumes, just keep the concentration constant.

http://www.public.iastate.edu/~pedro/rt_all.html
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Dear  Scottie -  I  had  the  same  problem  when  I  was  drawn  back  to
BigDyes,  Dilution  buffers  etc. Three  possible  answers

(1)  Feeling  masochistic -  use  Blue  Dextran  -possible  the  worst
chemical  in  the  world  to  weight

(2)  Purchase  PARAROSANILINE - I  understand  this is  the  dye  that
Amersham  use  well  it  is  pink  and  it  migrates  the  "wrong"  way.

(3)  Use  the  Perkin-Elmer  lane  Guides  -  just  been  REDUCED  in
price.  I  am  undergoing  psychiatric  help  on this - has  anyone  heard
of  Perkin-Elmer  reducing  anything ??  In  my  experience  the  lane
guides  work  very  well -  my  gels  track  automatically  now. One  dye
(odds)  is  Blue  dextran, the  second  dye  is  pink   nd  migrates  the
"wrong"  way  probably  pararosaniline. * They  contain  different tracking
oligo's - you  have  to  install  Data  Collection  2.6  to  use  the  5
dye  system - oligo's  are  orange. You  probably  know  all  this.

* pararosaniline  is  alo  called  Basic  Red 9.

Regards,
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Hi Scottie,

I use blue dextran at 25mg/ml. Works just fine. Make 50ml of loading dye
up with some of that and it will last a fair while.

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Hi,
ABI has a formula for loading buffer which I used on our 373 for years.
		deionized formamide
		25 mM EDTA(pH 8.0) with 50 mg/ml blue dextran
		5:1 ratio
I added a bit more blue dextran for darker color.
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Basic Red is OK.

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Hi,
We make our own loading dye based on the PE formula...as you probably know, it
is blue.  The composition is 25 mM EDTA, pH 8.0, containing 50mg/ml Blue
Dextran 2000.  I hope this is helpful.  Of course, if it is pink you
need..keep looking.
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The loading dye is available from Amersham.  Part number is US79448 and its
$32 per 1200ul.  They don't seem to want to give any details on the dye.
Good luck
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The recipe is:
1% pararosaniline-HCl (alias acid fuchsin) from Sigma dissolved in 90%
deionised formamide - 5 mM EDTA pH 6.5. Dissolve on a stirrer o/n then spin
10Krpm for 10 min on anything suitable and aliquot in Eppendorf tubes for
storage at -20 C.
We use it for both LiCor and ABI samples.
Hope it helps.
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>I dunno what the pink dye is in the Dynamic ET kits but I suppose you could
>always fall back on old faithful - Blue Dextran (also from Amersham
>Pharmacia Biotech) which has been recommended by ABI since the beginning.
>If you are really desperate I could always sell you a spare one... ;-)
>
>Cheers
>

Scottie Adams aka Pamela Scott Adams

Manager
Molecular Biology Core Facility
Trudeau Institute
100 Algonquin Avenue
Saranac Lake, NY    12983
Phone:  518-891-3080, Ext. 115
Fax:    518-891-5126
Email: sadams at northnet.org
sadams at trudeauinstitute.org
http://www.trudeauinstitute.org



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