cracking gel problem - (Jul/14/2009 )

KINDLY HELP!
I am performing a cracking gel to confirm the presence of my insert in the vector .I gel purified my insert of 4kb and cloned it into vector of 11.2 kb and by doing a cracking gel i am supposed to see a shift of 4kb(11.2+4kb insert ).


I am also working on another clone -the miniprep concentration seem to be high 183ng/ul,167ng/ul when i perform restriction analysis to check for the presence of my insert - i am expected to see vector band 6.5kb ,insert band 1.1kb 5 of the minipreps show bands at 8kb and 3.3kb and one miniprep shows a band at 3.5kb and 6kb the procedure i have used:
I pcr amplified the gene and after purification (promega pcr clean up kit) concentration was very low 13ng/ul restricted digested both my vector and insert with Asc1& Spe1 phenol choloroform extracted and ethanol precipitated both vector and insert ligated 1:3(vector:insert)ratio overnight and transformed I saw plenty of colonies but when i check none have the insert
Thank you very much in advance kindly help me solve this problem

Cloning sucks doesn't it? It is so easy in theory...

Firstly with cracking gels: you only need a little bacteria. I usually do a streak onto a stock plate, then put the tip/toothpick in 100ul of cracking buffer, then run 10-15ul of this on a gel. Remember cracking buffer does not have glycerol in it so you cannot have running buffer over the top of your gel, only fill up the rig so the buffer touches the sides of the gel, but does not go over it. If you want my protocol I can provide it.

Re: expected sizes - are you certain the vector sequence you have been given is correct? We have had vectors donated to us, and the vector maps have been incorrect (VERY incorrect). Love that they were donated but you really need to double check EVERYTHING (even things bought from Promega, Invitrogen, etc) before you start - what do your empty vectors look like after a digest??

For gel extractions: I find the Promeag SV kit variable - I prefer the MoBio kit myself, but everyone else in my lab uses the Promega one... try and get your hands on another brand if you're worried. I don't bother with phenol/cloro extracting the DNA, you run the rick of losing too much of it, or leaving EtOH in it, try ligating straight from the purified bands.

What was your selection agent??

-A.