Cells grow on Amp plates but not in LB with Amp - Amp problem, bug strain problem, or ligation problem ? or all of the a (Mar/27/2007 )
Hi all:
Have been trying to clone a 4.2kb fragment into a lenti backbone (9 kb) with amp resistance gene for the past two weeks. This was done via sticky- end digestion with two different REs and both appropriate cut fragments of vector and insert were gel purified using Qiagen kit. I did not dephosphorylate the vector and tried ligation at 1:1, 3:1, 5:1 insert to vector ratio at 16C for 2 hr. Electroporation into DH10B cells from invitrogen was fine. Tons of colonies were generated on 50µg/ml AMP plates which were made 2 months ago. However, only 5% of colonies grew in 100µg/ml AMP-LB and only 80% grew in 50µg/ml AMP-LB. Obviously, all grew in plain LB. Minipreps of the colonies that grew in 50µg/ml and 100µg/ml AMP were neg for plasmid DNA just like the ones that grew in in LB (no selection pressure to keep plasmid). Well, I thought the AMP plates were bad and the colonies that grew, even though they look robust and I delibrately avoided the satellite colonies, were in fact AMP- sensitive and when died when exposed to fresh AMP in LB. This is consistent with the absence of plasmid DNA in minipreps from the bugs that grew in 50- 100µg/ml AMP.
When I used freshly prepared AMP- plates (50µg/ml) from the next lab, I got much cleaner colonies which appear to confirm the theory that the AMP in my plates were bad. However, nothing grew in AMP-LB (100µg/ml) using the AMP stock from this lab. I think something more sinister is going on likely related to the bug strain or ligation gone bad.
The control lenti plasmid grew and I did get colonies from my linearized (with the two REs) but not religated backbone control. The former grew nicely in AMP- LB and the latter did not.
I have just ordered Carbenicillin (don't think it will make much difference) and Stbl4 cells from Invitrogen. I am also redoing my ligation.
I trolled the forum for the past two weeks and only came across one post describing a similar problem. The guy solved his problem by using new plates (which I used) and a diff kind of E coli strain.
I have tried newly made LB plates with Amp, newly made LB. I am currently streaking the colonies that grew in the original 50µg/ml AMP plate into other 50µg/ml AMP plates and plan to do minipreps on them to see whetehr the plasmid is still present. I am also growing additional O/N 5ml cultures using 50µg/ml of AMP instead of 100µg/ml.
I think I have proved that my AMP lab stock is probably bad but there appears to be other factors involved. The insert could be toxic and I am contemplating growing the plates/minipreps at 30°C but don't understand why robust colonies appeared in the AMP plates at the first place.
Any suggestions would be appreciated. I am sure there must be something simple that I missed.
The fact that a) you couldn't find anything in your minipreps, and 
your digested and non religated control produced colonies says that the vector was not in fact totally digested. To check for complete digestion, try ligating a bit of your purified vector and run a gel. If the majority of DNA is not much greater than 2x the length of the vector, you have incomplete digestion. To make sure the ligation itself is working, try ligating some of your marker DNA and run it.
your digested and non religated control produced colonies says that the vector was not in fact totally digested. To check for complete digestion, try ligating a bit of your purified vector and run a gel. If the majority of DNA is not much greater than 2x the length of the vector, you have incomplete digestion. To make sure the ligation itself is working, try ligating some of your marker DNA and run it.Thanks. I was hoping to just miniprep/colony PCR screen my way out of the it but I was not possible when my minipreps were negative ! (again, miniprep from control backbone was good). I was warned before hand that the RE digest with XhoI and NheI would be tricky and there would partially digested contaminants (The dude even said to use two units of each enzyme in my 10µl rxn mixture). How do you explain for the appearance of colonies in the initial Amp50 plates ? - I checked the plates (AMP 50) and minipreps (AMP 50) that I streaked out the original colonies this AM and none grew from the original plate so I guess the plasmid is lost.
I will check for the percentage of partially digested vectors today and will do another round of vector and insert DNA preparation this week- . Any other suggestions ?
not sure, but seems like something happen when joining your media with Amp...a reaction which reduce cell number by forming a toxic media for these cells 
I had similar problems with the pLenti6 vector from Invitrogen. Often I had no growth in most of my mini preps. I switched to 2X LB broth + amp and saw a huge improvement. Check the Invitrogen FAQ, they have lots of helpful tips for working with Lenti plasmids.
2 months is too long to use Amp plates reliably. They may work, they may not. In this case, I would guess that the Amp has broken down, so you're essentially using LB plates. They must have been well-sealed, because I have seen 2 month old plates with the consistency of plastic discs!
2 months is too long to use Amp plates reliably. They may work, they may not. In this case, I would guess that the Amp has broken down, so you're essentially using LB plates. They must have been well-sealed, because I have seen 2 month old plates with the consistency of plastic discs!
The 2 month old AMP plates were used at the very beginning of the experiment and freshly made (same day) AMP plates as well as AMP plates from another lab were used subsequently. In addition, new AMP stock was borrowed from another lab. Now that I am seeing simlar problems with fresh AMP- the issue most likely lies with ligation and just E coli with junk showing up. I have heard of using 2X LB with difficult vectors (lenti) i.e. to successfully grow them in minipreps- does anybody else have similar outcome with 2X LB ? I was also warned that the bugs might kick out the plasmid with the lenti- backbone if I leave my miniprep for too long (>24hr) but this is ridiculous- no growth! I always check them at 16hr (and them every couple of hours after that) after shaking in the INFORS at 37°C.
Have been trying to clone a 4.2kb fragment into a lenti backbone (9 kb) with amp resistance gene for the past two weeks. This was done via sticky- end digestion with two different REs and both appropriate cut fragments of vector and insert were gel purified using Qiagen kit. I did not dephosphorylate the vector and tried ligation at 1:1, 3:1, 5:1 insert to vector ratio at 16C for 2 hr. Electroporation into DH10B cells from invitrogen was fine. Tons of colonies were generated on 50µg/ml AMP plates which were made 2 months ago. However, only 5% of colonies grew in 100µg/ml AMP-LB and only 80% grew in 50µg/ml AMP-LB. Obviously, all grew in plain LB. Minipreps of the colonies that grew in 50µg/ml and 100µg/ml AMP were neg for plasmid DNA just like the ones that grew in in LB (no selection pressure to keep plasmid). Well, I thought the AMP plates were bad and the colonies that grew, even though they look robust and I delibrately avoided the satellite colonies, were in fact AMP- sensitive and when died when exposed to fresh AMP in LB. This is consistent with the absence of plasmid DNA in minipreps from the bugs that grew in 50- 100µg/ml AMP.
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I see the same problem as you. I made a construct using pcMV-BD which is kan resistance as vector, and PCR product of 1.7kb as insert. While all things ran smoothly and I got tons of colonies on my kan plates. However no colony grew in the kan-LB. I tried many times with different kan working concentration and changed almost all conditions-I borrowed kan and LB from the near lab also. Still I can not grow colonies in the LB, even in the LB without any antibiotic. I was puzzled. The cloning system was ok in our lab because it did work for the other guys. I made colony PCR and found my inserts were there! The PCR product as I knew had no toxicity. What's the problem?