PGEM-T easy gel - partial digestion? - (Mar/17/2008 )
Hello guys! Need your opinion on something, more of a confirmation really:
I cloned some PCR products into PGEM-T Easy, grew the bacteria and mini-preped with the Qiagen kit. I digested for 10 min with EcoRI to release the insert and run 1 ug of the digested product on a gel. The reason I digested for such a short time is that NEB claims that EcoRI can digest 1ug DNA in 5 min. Also, I didn't heat inactivate the endonucease.
I attach a picture of the gel. The first 3 lanes correspond to the first insert of ~750bp, the second 3 lanes to an insert of ~550bp, and the last two lanes are of an insert ~550bp long. Ladder --> https://catalog.invitrogen.com/productImages/2900/2852.JPG (left one)
All the inserts look really good, right size and exactly as they were after PCR amplification. My only question is about the many vector backbone bands, especially the one just below 4 kb. I think it's clear there is some undigested plasmid in different forms, which I attempted to link to the gel.
I am thinking the ~4kb band could be a vector digested only once, thus containing the backbone (3015bp) plus the inserts (750 or 550 bp). Especially since I only digested for 10 min. Do you think this is correct? Should I go ahead with my sequencing/in vitro transcription of the inserts?
Thanks in advance 
I cloned some PCR products into PGEM-T Easy, grew the bacteria and mini-preped with the Qiagen kit. I digested for 10 min with EcoRI to release the insert and run 1 ug of the digested product on a gel. The reason I digested for such a short time is that NEB claims that EcoRI can digest 1ug DNA in 5 min. Also, I didn't heat inactivate the endonucease.
I attach a picture of the gel. The first 3 lanes correspond to the first insert of ~750bp, the second 3 lanes to an insert of ~550bp, and the last two lanes are of an insert ~550bp long. Ladder --> https://catalog.invitrogen.com/productImages/2900/2852.JPG (left one)
All the inserts look really good, right size and exactly as they were after PCR amplification. My only question is about the many vector backbone bands, especially the one just below 4 kb. I think it's clear there is some undigested plasmid in different forms, which I attempted to link to the gel.
I am thinking the ~4kb band could be a vector digested only once, thus containing the backbone (3015bp) plus the inserts (750 or 550 bp). Especially since I only digested for 10 min. Do you think this is correct? Should I go ahead with my sequencing/in vitro transcription of the inserts?
Thanks in advance
Have you tried running a little bit of supercoiled pGem-T along side to check which band is of certain to be the supercoiled species?
If you have the time, why not try digesting one of your clones, about 100-200ng with 2U of EcoRI for an hour and run a half on the gel. Then return the next day and run the remainder. If your extra bands are missing, why not just try sending them in for sequencing?
well seems sizes are ok and the upper are undigested. But if you're concerned, digest 30' 1h and 2h (stop reaction by putting tube on ice, and load all 6 tubes alongside with undigested vector. You'll see if you have your clones.
By the way, if i digest 10' only, i expect to get undigested or single digested vectors.