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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.  :-)



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.

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

Look under protocols; look at 377 protocol:
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.

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.

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.

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.

Basic Red is OK.

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.
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
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.
>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... ;-)

Scottie Adams aka Pamela Scott Adams

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


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