Just a couple of things you might want to try:

1. Can you amplify by PCR your insert from miniprep DNA confirming that the insert is there ?

2. Have you performed a double digestion to liberate your cloned insert ? Have you tried just cutting with one such enzyme and demonstrating in those clones where you can detect the insert by PCR from miniprep DNA (as well as the initially colony hybridisation) that the single cut linear fragment is larger than the same fragment from a clone where you cannot detect the insert from miniprep by PCR, i.e. the first is vector + insert and the second vector alone and therefore as a single cut linear fragment is 1.1kb smaller

3. If you did perform a double digestion, how compatible are your restriction digestion buffers ?: For buffers that differ in terms of composition,typically I used to perform a single digestion for 2hrs (~ 500ng); Purify by phenol chloroform extraction and ethanol precipitation - or alternatively using a DNA purification column; resuspend the pellet and then re digest with the 2nd enzyme plus other buffer before running on an agarose gel

Thanks Laurence!

1. I have already thought of doing the PCR with the miniprep DNA.

2. I have done double digestion with compatible buffer for both the enzymes. I'll try and do the single digestion as well.

3. I have used the same buffer as I have used for double digestion of my vector and insert under all same conditions. So, I think, double digestion shouldn't have been a problem in this case.

Hi Tias

I routinely screen for correct recombinant inserts initially by PCR

That is because PCR can be more robust than Restriction digestion: Sometimes PCR will work with miniprep DNA that is not amenable to restriction digestion: That is because of contaminants that inhibit digestion or sometimes when the plasmid DNA is supercoiled. The latter can be caused by incubating with the NaOH containing buffer (#2) for longer than 2 min. I tend to be very strict about timing at this point in the procedure

Hope that helps !!