Help!!!!!! Ligation

Rodney Earl Pettway pettway at cygnus.tamu.edu
Mon Jun 10 15:38:43 EST 1996


I have a "HUGE" problem.  I have tried about 8 times to clone fungal
mtDNA PstI fragements into a pUC18 vector.

I set up the experiment in the manner:
The mtDNA was CsCl spun 3 times.
(1.) I digest 200 ng of the fungal mtDNA with PstI overnight.  I also
digested 10 ng of pUC18 with PstI overnight.  I know that this is not a
lot of DNA but I have used more DNA with no success.
(2.) I ran a gel with both to make sure that they both cut.
(3.) I increased the volume of both the cut mtDNA and the pUC18 to
500 ul and then phenol extracted them followd by a chloroform
extaction.
(4.) I ethanol precipitated the mtDNA and the pUC18 overnight.
(5.) I treated the mtDNA with RNase I for one hour.
(6.) To a microfuge tube I added 10 ng of pUC18, 200 ng of fungal
mtDNA, 12.75 ul of water, 2 ul of buffer and 1 ul of ligase (the stock
concentration of the ligase was 400,000 units/ml.)
(7.) I also set up a ligation reaction that only had digested pUC18 in the
ligation reaction.
(8.) I also had other controls for transformation frequency.

I saw no white colonies that had been transformed with my mtDNA/
pUC18 ligation.  I did not even see any blue colonies on these plates.

I did see 1,000's of colonies that had been transformed with the
pUC18/pUC18 ligation reaction.

>From what I can tell is that the mtDNA pUC18 ligation did not work.

The 8 other times that I did this I got 100's of white colonies but none
of them had mtDNA inserts.  They did have inserts but none where the
PstI inserts that I was looking for.

Please HELP me!!!!!!!!!!!!!!
-- 
Rodney Earl Pettway
Texas A&M University
Department of Plant Pathology and Microbiolgy
Intercollegiate Genetics
Program for the Biology of Filamentous Fungi
College Station, Texas 77843-2132
Email: pettway at cygnus.tamu.edu
Phone: 409-845-7614
Fax: 409-845-6483



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