big dye dilution

Harold G. Hills hhills at iastate.edu
Thu Feb 26 12:50:51 EST 1998


Apparently I was not very clear in my first bigdye dilution message.

The bigdye dilution buffer is meant to be used at 2.5X.  PE-ABD supplies it
at 5X.  Dilute the 5X with water to 2.5X.

Then replace the BigDye premix with the buffer.

4 ul BD with 4 ul of 2.5X buffer
2 ul BD with 6 ul of 2.5X
1 ul BD with 7 ul of 2.5X

I did 1 ul of BD with 7 ul of 5X buffer and also 1 ul of BD with 7 ul of
2.5X buffer.  The 1 ul with 2.5X buffer is clearly better than the 1 ul
with 5X buffer.

Ones needs to cut back on template and primer to achieve the best results.

I got very good results with 1 ul BD and 7 ul of 2.5X buffer when I used
0.25 ug DNA and 3.2 pm of primer.  We have traditionally used extra primer
because we get such a variation of actual primer when clients send it at
5pm/ul.

I hope this clarifys my earlier message.

Bruce Roe's group at Okalahoma has gone much further with the dilution.

Hal


Harold G. Hills, Ph.D. DNA Sequencing Specialist	515 294-9585
1184 Molecular Biology Building			    FAX 515 294-1597
Iowa State University                                   hhills at iastate.edu
Ames, IA 50011-3260
http://www.biotech.iastate.edu/Facilities/DSSF/ABRF   For ABRF 97 DNA
sequencing tutorial.
http://www.biotech.iastate.edu/Facilities/DSSF   For info about facility.





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