If anyone can suggest if I can use a lab made sequencing buffer of (400 mM Tris-HCL, 10mM MgCl2 and pH 9) in place commercial Sequecing buffer while performing Cyclic sequencing. How does the efficiency of the sequencing results varies?
This buffer will work with ABI big dye mixes and these diluting buffers and even just water have been successfully used to dilute sequencing mixes for many years successfully. In theory changing the relative amounts of Mg and Mn in a sequencing mix might change the profile of the sequence peaks so that the best sequence is at slightly higher molecular weights but I have never seen this in practice. The attached file is informative
Thanks Paul Rutland . I am convinced by what you said. I will try both water and also the recipe mentioned here.
Yes you can
I assume that buffer is 5x
Just keep in mind though that commercial buffers will sometimes have additional additives to help navigate tricky regions of sequence but the essence of the buffer for big dye sequencing is just what you have said
Make sure if you make the buffer to use molecular grade or HPLC water
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