Problems cloning into pET Kanamycin resistance vectors - (Nov/24/2005 )
Dear all,
my group are having problems cloning into pET vectors with kanamycin resistance gene. We have tried two vectors without any success. This is how it goes.
We have cut the vectors and inserts (sizes between 800 bp and 1500 bp) with NdeI / XhoI and get the right digestion products (digestion with pET42a removes the GST fusion and this is clearly visible on the gel).
Gel purify the vector (Qiagen). Look at DNA amounts on gel after isolation to estimate amounts for ligations.
Setup ligations with T4 DNA ligase (both fast link and standard T4)
Electroporate DH5-alpha and/or BL21 (DE3) Gold (transformation efficiencies 10^8 to 10^9).
Recover for 1 hr at 37degC in SOC medium. Plate 1/3 total volume (usually 200 ul) on Kan plate.
Get no colonies on Kan plates (25 microg/ml)! Even if the double digest appears to be successful, we always get a couple of colonies on the control plate (cut vector only) but nothing grows. Transformation with the uncut vector is fine and we get colonies on Kan plates.
We have even tried a different gene that has been successfully cloned into pET 24 (an Amp vector) and this gene gives NO colonies back.
We need to clone into a kan plasmid as our genes of interest are beta-lactamases.
I am sure that this is something very trivial that we are not doing but cannot for the life of me figure it out. We have no problems cloning into Amp pET vectors, just the Kan ones. I would be grateful of any suggestions.
Thanks,
Dafydd.
Someone in my lab had similar problems, then he changed from SOC to LB medium and got clones... no idea why...maybe try it
Stardust
What you've outlined appears reasonable. Do you know the Kan concentration in you plates is correct? Is the one hour non-selective incubation done with agitation? Do you know your cells are competent?
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How are you generating your insert? If it's by PCR with restriction sites added to the 5' end of the primers, how many irrelevant 5' bases did you include? As I recall, Nde I is particularly stubborn to digestion by 5' primer addition -- I routinely TA clone Nde I ended PCR products as an intermedite step, and recover the insert from the TA vector.
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We are pretty sure the plates are at 25 ug/ml kan. Nothing grows on them in the absence of a kan-r plasmid. We do shake the cells after the addition of SOC media. We are using Stardust suggestion and using LB. We get through a lot of competent cells (a batch every month) and test the competency every time with pUC19 so we know that is not a problem. The uncut pET kan vector transform OK in the same cells.
The insert is generated by PCR with the NdeI site 7 bp from the end. This is generally works fine for us. We have tried to clone another unrelated gene that ligates nicely between the NdeI and XhoI sites of an Amp pET vector, but even this ligation failed.
Are next strategy is to induce the beta-lactamase genes in the BL21 cells prior to plating them on Amp plates to see if get any colonies. Also, as well as the suggestion by Stardust, we are trying the good old fashion chemical transformations.
Thanks for the suggestions.
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Hi,
Right from pcr amplification of your insert and vector purification by mini prep, you avoid running on gel simply purify after every digestion with pcr purification kit. I sure hope you will get succeded.
For me after 3months of enormous investigation reproducibly succeeded. All the best.
Amadhy
Hello !
I have the same problem with pet vector and ptriex... both from novagen and i think they are from the same original vector.
I'm not able to clone Nde/Xho, Nco/Xho1, Xho1/Xho1, Nco1/Nco1 or blunt via sma1...... att he same time i'm doing a ligation control with my insert blunt with puc19 linearized blunt... and it work in puc19 and not in pet !
Where you able to clone ? What was your problem ?
Any other sugestion ?
Dear all!
I got the same problem by using pET-28a!!
I amplify a 4.5kb fragment and sub-clone it by using the TA cloning kit, then cut the fragment by NheI/HindIII (NEB).
In the other hand, I digested the vector by NheI/HindIII (NEB).
After digestion, I separated them by agarose gel, and elute the fragment and the vector by gel and PCR clean up kit (Promega).
After the elution, ligated the insert to the vector by T4 DNA ligase (NEB) in ratio 1:7, incubate at 16 degree C overnight, followed by transform into chemocompetent cell (TOP10).
After overnight incubation in 37 degree C, no colony formed on the plate with 50ug/mL kanamycin.
I feel very frustrated because I have repect this process (PCR-->digestion-->gel elution-->ligation-->transformation-->plate cells) for many times by different variations (e.g. overnight ligation at room temp.; sequential digestion of the fragment; ligate in 1:3, 1:9 ratio; etc),however still no colony formed on the plate.
I just wonder, is it the problem with the NheI site? Because it is locate in front of the T7 tag, would it be THE problem, so I can't ligate a fragment there?
Please help!!! 
I got the same problem by using pET-28a!!
I amplify a 4.5kb fragment and sub-clone it by using the TA cloning kit, then cut the fragment by NheI/HindIII (NEB).
In the other hand, I digested the vector by NheI/HindIII (NEB).
After digestion, I separated them by agarose gel, and elute the fragment and the vector by gel and PCR clean up kit (Promega).
After the elution, ligated the insert to the vector by T4 DNA ligase (NEB) in ratio 1:7, incubate at 16 degree C overnight, followed by transform into chemocompetent cell (TOP10).
After overnight incubation in 37 degree C, no colony formed on the plate with 50ug/mL kanamycin.
I feel very frustrated because I have repect this process (PCR-->digestion-->gel elution-->ligation-->transformation-->plate cells) for many times by different variations (e.g. overnight ligation at room temp.; sequential digestion of the fragment; ligate in 1:3, 1:9 ratio; etc),however still no colony formed on the plate.
I just wonder, is it the problem with the NheI site? Because it is locate in front of the T7 tag, would it be THE problem, so I can't ligate a fragment there?
Please help!!!
I am just wondering if your T-cloning was succeed.If it is, the Nhe should be ok.
And my suggestion, try to digest your vector for longer time. For some instance, NheI got a low efficiency.