Interpreting RE digest results after blunt cloning - (Feb/09/2006 )
I need some help interpreting the results of my restriction digest. I have a 2.7 kb blunt-end PCR fragment which I put into vector using Invitrogen's ZeroBlunt vector. The vector is 3.5 kb, and has EcoRI sites flanking the insertion site. It looked like the ligation worked, in that I got lots of colonies. However, when I digested the extracted plasmid with EcoRI only half of the colonies gave me the bands at 2.5 kb and 3.5 kb I expected. THe others all gave bands at 5.5 kb and 10 kb. Can anyone tell me what is possibly causing these large bands? I'm not findig any pattern related to insert source or plasmid purification.
well, I am no expert, don't get me wrong...but often during cloning you get some random junk transformants. I have never sequenced any and don't know what they are; I have always thought they were likely to be due to strange concatamerization events during ligation, otherwise known as 'DNA from outer space'
10kb = 3x your vector; 5kb= 2X your insert
so, I would guess concatamerization during the ligation. But if I were you, I would be very glad to get the right clone and who cares about the other stuff?
although it is certainly a matter of curiosity...can anyone else shed any light?