DNA Sequence Pile-ups

Mark Guiltinan Mark_Guiltinan at AGCS.CAS.PSU.EDU
Mon Jun 21 19:21:02 EST 1993


Since there is so much interest in this topic, I will post our procedure used
for TdT "pileup" elimination.  After following the sequencase protocol, we
simply add the following steps;


Use terminal deoxy transferase to help eliminate "pauses" by Sequenase.  Add 1
ul TDT solution to each tube (G, A, T, C) after termination step, (at same
point one would normally add STOP), and incubate an additional 30 min., 37 C. 
Then add Stop. 
TDT Solution Recipe (10ul):
1 ul10 mM dATP
1 ul 10 mM dGTP
1 ul 10mM dCTP
1 ul 10 mM dTTP
1 ul 20 units/ul TDT
2 ul Sequenase Reaction B. (5x)
3 ul water


Mark Guiltinan
Penn State Biotechnology Institute
mjg at psupen.psu.edu



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