Cloning - PCR and Cloning problem (Jun/14/2010 )

I have a 10.5 kb vector in which I am introducing a mutation with PCR primers. I designed primers such that I have the required mutation and also a sticky end restriction enzyme site, AvrII.
I did the PCR, I ran 3 ul of the sample and saw the 10.5 kb band. I took 10ul of the PCR mix, put AvrII and DpnI. I gel extracted it.
Then I ligated o/n with T4 DNA ligase at 4C. I ran 5ul of this reaction on a gel. I still see a band of 10.5 kb.
Then I transformed it along with my original vector as positive control. I saw a gazillion colonies.
I grew 1 colony from my positive control and 8 of the experimental.
After mini-prep, I ran 5 ul of the reaction just to see if I had plasmid, but I saw nothing. I haven't gotten to checking the plasmid by restriction digestion becoz I see no plasmid on the gel. What might be going on?

Help!!!

may be the concentraion of the plasmid is too low to see on the gel?
try loading may be 20ul of the miniprepd plasmid

it happens to me some times also, that's why I always pick more colonies and grow in different broth and extract separately. no worries.....my advice to you pick another colony and regrow in broth.

also, I have noticed that some times when I load my sample on the first well of the gel it looks so faint. so next time load in the middle wells.

Thanks guys!!

Curtis on Jun 23 2010, 09:43 PM said: