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
I hope this clarifys my earlier message.
Bruce Roe's group at Okalahoma has gone much further with the dilution.
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
http://www.biotech.iastate.edu/Facilities/DSSF For info about facility.
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