too many cloning false positives - (Feb/24/2008 )
Hi,
I am fairly new to cloning and have been getting a lot of false positives (vector without insert, no ligase) after transformation. From my control, I find that I get many colonies from transforming the digested vector, anywhere from 10-200+. I typically take 10-20 uL of a miniprep of pET or pACYC based plasmid and double digest in 20-50 uL total volume, for 2 hrs. to over night. I have used miniprep kits from Qiagen or Zymo. I use enzymes from NEB or Fermentas. I typically use a set of enzymes, BamHI, EcoRI, NcoI, NotI, KpnI, PstI, NdeI, XhoI, BglII. After digest, I usually do not heat inactivate. I gel purify on 10 cm long minigels. Is that too short?
I have tried tricks like adding another restriction enzyme after ligation that cuts in the MCS site that should be replaced by the insert. That sometimes helps. It seems like there is some undigestible plasmid which is still capable of transforming cells, yet which I can't separate by gel purification.
I am fairly new to cloning and have been getting a lot of false positives (vector without insert, no ligase) after transformation. From my control, I find that I get many colonies from transforming the digested vector, anywhere from 10-200+. I typically take 10-20 uL of a miniprep of pET or pACYC based plasmid and double digest in 20-50 uL total volume, for 2 hrs. to over night. I have used miniprep kits from Qiagen or Zymo. I use enzymes from NEB or Fermentas. I typically use a set of enzymes, BamHI, EcoRI, NcoI, NotI, KpnI, PstI, NdeI, XhoI, BglII. After digest, I usually do not heat inactivate. I gel purify on 10 cm long minigels. Is that too short?
I have tried tricks like adding another restriction enzyme after ligation that cuts in the MCS site that should be replaced by the insert. That sometimes helps. It seems like there is some undigestible plasmid which is still capable of transforming cells, yet which I can't separate by gel purification.
Hi,
It is probably your double digestion which is not working well. Your vector may be digested by one of the enzymes and not by the other because of several reasons. There may not be enough space after your first digestion in order for your second enzyme to work on your vector.
Instead of double digestion you may try sequential digestion. Just digest the DNA with your first enzyme, purify the fragment (get rid off the enzyme), digest with your second enzyme.
Are you sure both of your enzymes are working? Does each of them linearize your vector? Are all your enzymes happy with double digestion conditions (temperature, pH)? They may not be working efficiently in the same buffer and temperature when double digesting...
If you're getting false positives from plates without any ligase added, then it is probably from undigested vector. You could try running your gel fragments out on a longer gel to get better separation from the vector DNA. If the problem is that you think the double digest might not be working well - i.e. not cutting twice, you can try doing the digests separately like it was suggested, or you can try to phosphatase the ends of your vector. If it was only cut by one enzyme, then this will reduce the number of plasmids that recircularize.
As others suggested, the vector is not being digested properly. may be the enzyme are not digesting or some may b buffer problems.
Eg. Take bamH1 and EcoRI and digest them and see how if there is background colonies. Both are good cutters and work efficiently in 1-2 hrs. Also digest the vector with only one enzyme separately and run it on gel to check if the enzyme is digesting or not.
Thanks for the suggestion. I've tried this before with BamHI (without gel purification after digest) and got lots of background colonies.
Eg. Take bamH1 and EcoRI and digest them and see how if there is background colonies. Both are good cutters and work efficiently in 1-2 hrs. Also digest the vector with only one enzyme separately and run it on gel to check if the enzyme is digesting or not.
If you ever want to test the efficiency of your restriction enzymes on your vector, digest the vector (in two separate reactions) with each of the cloning enzymes and another restriction enzyme at a nice distance away from the cloning site (say 2000 bp if you have a 7000 bp vector). Choose a reliable restriction enzyme. Here's the setup i'm talking about:
-digest A - EcoRI + RE x
-digest B - BamHI + RE x
Importantly, use the same conditions as your cloning digest - same buffer mainly.
If each digest goes fully to completion you should get 5000 + 2000 bp bands and any incomplete digestion will show up clearly as a 7000 bp band. This is a quick and very informative test to check that each of your restriction enzymes are working. I would give that a go.