Ligation/ transforamtion troubles - (Apr/18/2007 )
I have been trying to ligate in a 1448bp insert into a 7863 bp vector. I have many problems with this process. I have made new plates, I have tested the restriction enzymes and ordered new T4 ligase. I have also fiddled with the concentrations of the ligation mix. Here is one one my many problems: I used undigested vector in my transformation and they grew on my LB amp plates. However, when I went through the process with uncut plasmid (starting with gel purification) I did not cut the plasmid. I did do a mock ligation over night at 16C. I then did the electroporation to transform the cells. I plated 200ul on the LB Amp plates and incubated for 16-18 hrs. I did not get any colonies. I don't understand what might be happening. The plates 50mg/ul were used and nothing but at 100mg/ul I got colonies. Help please...I have no way of testing to see if my ligation worked if I can't get the postive control to work.
for your positive control (the mock experiment),
can you confirm (by gel) that there is DNA in the tube, immediately prior to transformation? What transformation method are you using, chemical transformation or electroporation. There is a good chance that you might have lost your DNA when you percipitated and 70% EtOH washed after the ligation step. How much DNA are you throwing into the ligation mix?
Adding dextran helps pull down DNA, and make the pellet easier to see.
"plates 50mg/ul were used and nothing but at 100mg/ul I got colonies"
How many colonies did you get? 100s and 1000s or just one or two. THis might just be a case of contamination.
Afraid I don't understand your positive control very well. As it is:
- You did not cut the plasmid
- But you still run it on a gel and did gel purification????
- Then after purification you ligated and transformed?
If that is right, one thing is I don't know how you cut your plasmid out. Uncut plasmid when run on gel should give you more than one band, right?
If you are doing positive control, how about digest with 1 enzymes, run gel, purify, ligate and transform?