Help!!! liagtion problem

Rodney Earl Pettway pettway at
Mon Jun 10 13:33:03 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 
(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.

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