Hello researchers,
Here's the thing. I've this expression kit (Champion™ pET101 Directional TOPO™ Expression Kit REF 45-0175) and I've read about the protocol provided by Invitrogen. Where it describes the exact protocol I've to follow in order to obtain positive colonies with my desired insert.
I designed the primers with the overhang sequence (CACC) and removed the stop codon of my gene, and perfomed the PCR to isolate the gene of interest (704 pb) that I want to clone in E. coli TOP 10 using this kit. The PCR was successful and then I had to follow the procedure of the kit, which consist in the ligation of the insert in the plasmid (pET101/D-TOPO), performing the TOPO® Cloning Reaction in Chemically Competent E. coli using:
1 uL of PCR product (40 ng/uL)
1uL of Salt Solution
1uL of TOPO vector (15-20 ng/uL)
3 uL of Sterile Water to complete a total volume of 6 uL.
I mentioned the concentration of the PCR product and TOPO vector because of the molar ratio, Invitrogen recommends 0.5:1 to 2:1 molar ratio of PCR product:TOPO vector.
Then I made the same but with no PCR product because I wanted to use it as a control in my transformation with E. coli TOP 10 (also keeping a final volume of 6 uL). To visualize later the different with a MiniPrep, PCR or something. And let the TOPO cloning reaction (with my PCR product) mixing gently and incubating it at room temperature (25 ºC).
After getting this TOPO cloning reaction ready I performed the appropiate protocol to transform the Chemically Competent E. coli TOP 10 (in the freezer -80ºC).
So, after performing this protocol of transformation I made the respective controls (I Used LB + Ampiciline (100 ug/mL) medium) and let them in the incubator (37ºC), their colonies yield was low. And the exact problem that I've with this kit is that I didn't visualize any colony with my insert!
What I did after letting the cells grow was isolate each one in another plate as a individual colony. I got randomly 9 colonies, and tried to perform a PCR colony with my primers of the gene of interest (result was negative) and maybe I thought that the DNA concentration was kind of arbitrary so I moved the colonies from their solid medium to a liquid medium with the same composition. And then I performed the MiniPrep and all of the colonies (included the control with NO PCR product, was successful) showed the same band size, nonetheless I performed other PCR with the primers of my gene of interest and once again my results were negative. The only conclusion of this procedure is to think about the ligation of the PCR product in the plasmid. Any advice? Anything would be great, THANK YOU SO MUCH!
Hello David,
The strategy you have attempted appears great except for just one step that is most likely the cause of your low transformation efficiency. Your problem is due to the molar ratio of insert to vector you are using. Because your insert is so small (704bp) compared to your vector (I believe these pET TOPO vectors are approximately 5kb), you need much less of you vector and more of your insert for your efficiency to increase. You cannot just double the mass you are adding for PCR product (40ng) to cector (15-20ng) since you need to take into account the moles of DNA present in the reaction. Another tip is that these reactions can often use up to 5:1 insert:vector for better efficiency if 2:1 isn't working. Use the following calculator for the amount of each DNA piece to use: http://nebiocalculator.neb.com/#!/ligation
Hope this helps!
Max
I've used the TOPO cloning system and I can tell you it has worked brilliantly and saves a lot of time but it can get pretty pricey. The only issues I ran into was when I didn't realize that the TOPO vector has a bunch of restriction sites in the MCS so I was accidentally cutting my insert three times, another time I forgot to use Taq polymerase so I didn't have the A/T overhang that the TOPO ligation enzyme requires. I agree with Max, I definitely used a healthy excess of insert when I was doing this work. Also, the TOPO incubation for ligation takes less than 5 minutes, just FYI.
i worked before with the same cells and vector, actually it gives very good results.
in your case firstly, i advise to obtain the primer again from another company. and try, if the same result, i think its due to efficiency of the TOPO enzyme
also check the pH of the LB medium, may be shifted
Good Luck.
Gamal
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