Sequencing of cosmids

jmv at canctr.mc.duke.edu jmv at canctr.mc.duke.edu
Fri Feb 12 18:12:56 EST 1993


In article <1993Feb10.124607.18120 at gserv1.dl.ac.uk>, fsteers at crc.ac.uk (Ms F.J. Steers) writes:
>
>Does anyone have any suggestions about sucessful sequencing of cosmids.  I have 
>been trying to sequence cosmids using the "Sequenase 2.0" kit and the DMSO 
>denaturing method and getting unreadable or no sequences.  I think that my 
>technique is OK, as I,ve recently used the same protocol on some plasmids
>and have been getting good results.

HI Fiona, 
	I've run into the same wall, determined that the concentration of
cosmid DNA required to duplicate the Sequenase 2.0 working conditions (ra-
tio of template to primer) is too high to be practicable, and discovered
cycle sequencing...the advantage being that one can start with very little
template DNA in a small-volume reaction.

	Kits that work well include Promega (comes with primer end-labeling
reagents PNK and PNK buffer at relatively low extra cost); Perkin-Elmer
Cetus (no PNK, but a pipetting step is eliminated by inclusion of polymer-
ase in the sequencing buffer); NEB and Stratagene, which I haven't tried, but
I've talked with tech reps who say cosmids can be done with their kits and have
sent me protocols and spec sheets which look almost exactly like the Promega
fmol kit's stuff, excepting PNK. The general protocol is 1. end-label the
primer (PNK and gamma-32P or 33P ATP), 2. combine cosmid DNA, labelled primer,
buffer and polymerase, 3. aliquot mixture to d/ddNTP mixes, 4. overlay with
mineral and throw into PCR machine, 5. add stop dye, denature and run on PAG.

	Here are conditions I've worked out especially for cosmids:
1. use 600 ng (about 22 fmol) cosmid DNA to 15 ng (about 0.3 pmol) primer,
 if your primer is ~20 bp
2. follow kit protocol for rest of reagents, doubling each (doubling reaction
 volume gives us more consistent results)(so for the kit protocol, 300 ng
 cosmid DNA per reaction, following exactly)
3. PCR machine should be set at: initial denaturing 95C, 3 min.; 20-30 cycles
 of 95C for 40 sec./ 50C-60C for 30 sec./ 70C for 1 min; end.
 (depending on sequence, if known, and primer sequence)
4. denature 70-75C for 3 min, run on 5% gel, expose ON at -80C.

	I'm getting good results with 33P label. However, if someone knows a
non-radioactive sequence method that is not as labor-intensive as Southerns
and works, please let me know.

good luck!!
	--DB
	---Duke U Med Ctr Neurology Div.



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