Cloning - how to avoid religation and no insert? - (Nov/14/2005 )
As a separate comment, I think that Phage's technique would be really useful for situations where the insert has to be in-frame with respect to particular restriction sites, for the purposes of cloning into one or more expression vectors later on. It may be a handy alternative to what I usually do, which is to ligate dsDNA oligo linkers, containing restriction sites, into a half-way house vector, and isolate clones containing the linker insert, and then clone my gene into the modified vector. Phage's technique would take one cloning step out of the whole process.
Hi again
This cloning is just about to drive me nuts...
An update:
I've managed to add 3'A-ends to my blunt-ended PCR fragment and clone this into the pCR 2.1-TOPO vector (not many clones but one out of 12 tested was positive). After an over night digestion I got 3 bands as expected when digesting with XmaI/BglII together (the vector itself has one BglII-site). I haven't sequenced my insert but since I got the expected fragments after this digestion I figured it confirms that the restriction sites are as they should be (maybe I should do some more restriction-site-mapping of this clone). I then purified my 2 kb fragment from the gel using Quiagen's PCR purification kit (eluted in elution buffer). The concentration was low (6 ng/µl measured on NanoDrop).
Then I ligated using Quick ligase (37 ng vector + 3 and 4 times molar excess of insert, incubated for 5 minutes at RT). Uncut vector gave plenty of colonies showing that the transformation itself worked, but I've got absolutely NO colonies with my insert
. Cut/purified vector didn't give any colonies either, which is fine. But I really can't understand why I don't get any colonies with my insert... I've even tried a completely new Quick ligase kit when I finally noticed that the ligation buffer could be sensitive for repeated freeze/thaw-cycles (I guess the ATP is somewhat unstable), but that didn't help at all.As far as I can understand, both my vector and my insert should be OK now. They are both purified from gel and should not be contaminated with any uncut DNA or attaching restriction enzymes. I did NOT heat inactivate my samples after digestion before running the gels - cold that be a problem?? (one of the enzymes can't be inactivated that way). I've always assumed that the enzymes were lost in the process of gel electrophoresis and the following purification of DNA cut from the gel - is it possible for some of the enzymes to still be in my samples...?
Is there any reason to believe that "regular" T4 DNA ligase used at 16oC should be a better choise for the ligation? I'll give that a try over the week-end unless someone gives me a better idea... Maybe I should start looking for another job...
Any input is greatly appreciated
Sonja
Many things can go wrong on a gel purification. 6 ng/ul is barely at the detection limit for the Nanodrop. I'd suggest that you have no DNA in your tube, or at least very very little. I'd suggest cutting larger amounts of your Topo cloned vector and running several wells in parallel. Use either no or longwave (365 nm) UV to image the gel slices. Short wavelengths can trash your DNA very quickly.
Solubilize the agarose in whatever volume it takes, and repeat the loading of your column several times to flow all of the sample through it. You might think about the Qiagen Minelute columns, which elute into small volumes. Make sure you have added EtOH to the wash buffer. Make sure you spin your washed samples a second time after emptying the collection tube. Heat your elution buffer. Let the elution buffer sit on the column for a few minutes. Elute with 10 ul (Minelute) or 30 ul (normal column). Check that you see the typical DNA hump, in addition accepting the number from the Nanodrop. Think about running a gel with your recovered sample to verify that you have the insert and that it is the correct concentration. You can't see less than about 20 ng on a gel, so load at least that much. How are you preping the vector? There could also be a problem there. Are you heat killing the enzyme (assuming it can be)? Are you adding too much ligation mix to competent cells? More than about 5% by volume is toxic in chemically competent cells. A gotcha we recently encountered was that the NEB EcoRI buffer has Triton-X100 in it, and enough was carried over through the ligation and transformation reaction to be quite toxic to the chemically competent cells.
You might consider not gel purifying your DNA at all, and simply mixing the DNA together, cutting, and religating. This would work well if you have a selection for the recombinants. Does your target vector have a different antibiotic other than Amp and Kan, which I believe the Topo plasmids carry?
Solubilize the agarose in whatever volume it takes, and repeat the loading of your column several times to flow all of the sample through it. You might think about the Qiagen Minelute columns, which elute into small volumes. Make sure you have added EtOH to the wash buffer. Make sure you spin your washed samples a second time after emptying the collection tube. Heat your elution buffer. Let the elution buffer sit on the column for a few minutes. Elute with 10 ul (Minelute) or 30 ul (normal column). Check that you see the typical DNA hump, in addition accepting the number from the Nanodrop. Think about running a gel with your recovered sample to verify that you have the insert and that it is the correct concentration. You can't see less than about 20 ng on a gel, so load at least that much. How are you preping the vector? There could also be a problem there. Are you heat killing the enzyme (assuming it can be)? Are you adding too much ligation mix to competent cells? More than about 5% by volume is toxic in chemically competent cells. A gotcha we recently encountered was that the NEB EcoRI buffer has Triton-X100 in it, and enough was carried over through the ligation and transformation reaction to be quite toxic to the chemically competent cells.
You might consider not gel purifying your DNA at all, and simply mixing the DNA together, cutting, and religating. This would work well if you have a selection for the recombinants. Does your target vector have a different antibiotic other than Amp and Kan, which I believe the Topo plasmids carry?
Hi Phage434
Thanks for being so helpful
You may be right about my insert; it could be absent... I will run a gel (I hesitated to do that since I have so little of it, but it's nothing to save for later experiments if it isn't there...). I did try to be careful when cutting it from the gel using preparative UV light for as short time as possible. Regarding the gel purification I'm sure the wash buffer contains EtOH and I made sure to spin the washed samples an extra time to remove all of the buffer. However, I did not heat the elution buffer (how much? 37oC?) but I left it on the column for about 1.5 min before spinning. And I used 30 µl elution buffer to try to concentrate the sample...
I have not heat killed the enzymes since one of them doesn't respond to this. I'll try that the next time so at least one of the two is inactivated.
And I use 4-5 µl of the 20 µl ligmix (Quick ligation from NEB) to 50 µl of heat-shock competent cells, which seems to be OK since I have a control-ligmix with uncut pGL3 vector that works very well.
The pGL3 vector has only Amp selection while the Topo-vector has Amp + Kan, so it will be difficult to select for recombinants if I skip the gel purification and just cut, mix and ligate. It will be possible to identify correct clones from those with inserted TOPO-vector fragments or religated TOPO-vector, but I'm afraid it could be many clones to pick before finding the one I need. But it's a possibility if gel purification fails again.
The pGL3 vector has been cut with XmaI, purified from gel, cut with BglII which cuts only a few bases away from the XmaI site (but many bases enough for the enzyme to do it's job) and purified using Qiagen's PCR purification kit. It looks nice on gel and since it doesn't religate in my experiments I believe both enzymes has cut the way I want them to. So the vector receiving my insert should be OK, I hope.
I'll start with checking my insert on a gel and grow more of my TOPO-clone.
Thanks again
Sonja
Hi everyone
hi sonja1
i heve experienced the same problems that you describe-
i think that the overhangs added to pcr primers with cutsites recommended by for example
neb are not always sufficient!
since the i mostly use topo ta clong (TOPO pcr 2.1- vector from invitrogen)
i use the protocol that fred_33 recommended-
after pcr amplification with pfu add 1µl taq-polym. for 10min at 72°
after that that 1-3µl to your top recation-
( in the instruction they recommend to use 1µl topo vector-
i ofund it to work grat if u use only 0.5µl topo vector- that way u can make this expensive kit last twice as long!!)
if u use a plasmid as a template for your pcr with eg amp resistance make sure to plate your clong transformation on kanamycin -plate since the topo-vector has both amp and kana-resistence
that way u avoid transformation of your plasmid!
the topo pcr2.1 has a lacZ gene wich is destroid during clong when an insert is luated into the vector- so u can put x-gal on your plate
religated empty vector will show up as blue colonies - your positive clones will be white-
the efficiency when picking only white ones is about 90% positves ( due to my experience ;-) )
i always do midi prep of the positive topo clone to make sure i have enough insert after restriction digest and following gel purification!
its a lot more work compared to just clone pcr fragments-
bur with this i make sure that i have cut pcr fragment with right cut sites
and usually further cloning into desired vector works pretty well!
sorry for writing so much -
hope i could help as a newbie here!
cheer flx