only one band after double enzyme digestion - (Mar/10/2008 )
HI, Dear ALL:
I did a TA cloning and digest the miniprep with two different enzyme. when i run electrophoresis, 3 of them have the vector band, the others (9) have the insert band, but none of them has both bands.
what is the reason? i can understand the only upper band ( vector) may be caused by self-ligation, but why only one lower band.
can you give me some suggestions?
TX.
A guess -- only one of your enzymes is cutting, and you see vector+insert bands in some, and vector only in others. Are you super-double sure of the lengths? Run some parent vector (cut) on the same gel. Try cutting with each of the enzymes separately.
I suggest you to check the buffer.
Is the activity of both enzymes in that buffer is good enough?
Or sometimes enzymes get old. try using new one.
are you sure that the 2 enzymes don't cut to any other site producing equal size bands.
also check the context of your enzyme, some enzymes like Xba are sensitive to dam methlyation, when the dam recognition sequence overlaps the restriction site.
I did a TA cloning and digest the miniprep with two different enzyme. when i run electrophoresis, 3 of them have the vector band, the others (9) have the insert band, but none of them has both bands.
what is the reason? i can understand the only upper band ( vector) may be caused by self-ligation, but why only one lower band.
can you give me some suggestions?
TX.
The insert band only after a miniprep sounds strange. Are you sure it's the insert band? Sometimes i find every now and then with my minipreps i get a single medium size band in the restriction screen. I'm not sure what this is but I'm sure it is isn't the insert.