simple method for sequencing from boiling mini-prep

Chuck Miller rellim at MAILHOST.TCS.TULANE.EDU
Mon Aug 8 10:53:10 EST 1994


In an earlier sm010b at uhura.cc.rochester.edu (Sean) asked for a simple
method for sequencing from mini-prep DNA.

I use the boiling lysis method of Holmes and Quigley (Anal. Biochem.
114:193) to prepare the supercoiled plasmid. Using an endA strain of E.
coli --like DH5a-- eliminates the need to do any DNA clean up (no RNase,
phenol, glass milk, etc.) after pelleting out the cell debris and ppt'g.
the DNA with isopropanol.
 
-Resuspend the DNA pellet in 50 ul TE.

-Place 18 ul DNA (about 1-4 ug) in tube and denature for 5-10 min by adding
2 ul of a -2M NaOH, 2mM EDTA soln.

-Neutralize with 2 ul of 2M ammonium acetate pH4.6

-add 100 ul EtOH, ppt. quickly

-resuspend in a small volume according to your favorite sequencing protocol
with an -approximately equal molecular primer:template ratio. Both
Sequenase and Promega -Taq-track protocols work well at this point. I get
best results by primer extension -labeling by incorporating a hot
alpha-labeled dNTP. If you are new to sequencing, I -suggest you start with
a sequencing kit and evolve your own favorite method.  
-usually I get 200+ bases of seq. info. when using the method above.
Dr. Charles A. Miller 
Dept. Environmental Health Sciences  (374 CBR)
School of Public Health
Tulane University Medical Center  
1501 Canal St. Box SL29                 
New Orleans, LA 70112               
Ph. 504-585-6942                          
Fx. 504-585-6939
rellim at mailhost.tcs.tulane.edu                                             
                          
                   






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