Strange result after ligation and transformation - (Dec/19/2006 )

Hello everybody in the www
My resent ligation troubles are:
PCR products (300 bp, 700 bp and 1350 bp) 3' blunt (no digestion) 5' EcoRI digested (after MinElute purification from Qiagen)
After digestion, purified again
pGEX-Vector (5000 bp) first digested with SmaI
gel purified
than digested with EcoRI for >4h
again gel purified (this was the last trial)
ligated o/n @ 4°C (ratio 1:5)
transformed ligation with insert, without insert and unligated opened vector
everything looked wonderful: 1 colony on the negative controls and 19(700bp), 29 (300 bp) and 103 (1350bp) colonies for the ligations
miniprep of the o/n cultures (for each cloning 5x)
control digestion with EcoRI
for the short fragments I got (if i was really lucky) 1 clone containing the insert and for the 1350 bp fragment until now, I didn't get a single clone containing the insert.
How come, that I have a grate transformation (it even seemed to be a good ligation) and at the end I can't find the clones containing the insert?!?!java script:emoticon(':blink:', 'smid_7')
Hope, that somebody has THE IDEA!!
Cheers
Mue

Did u try running the control vector and ur mini prep DNA - all of them undigested to verify if there is an increase in size of the minipreps DNA. Its a good indicator if u have an insert or not than to try digesting it out.

Also abt the control, do u add ligase to the undigested vector?

Hi,

No great idea. Do PCR screening on your colonies, since you have primers ready.

Bests