Dear experts in molecular biology,
I am having trouble cloning my PCR-amplified ~4.4kb fragment into the pENTR/D-TOPO vector (2.58kb) directional cloning vector from Invitrogen. I am using a protocol from the manual of the TOPO cloning kit.
After amplification of my fragment from the template plasmid using properly designed PCR primers, I have a very strong single blunt-end PCR product that appear to be about 4.4kb in size, with any faint secondary bands on a 1% agarose gel. According to the manual they don't require obligatory purification of the pcr product if you have only one band. Therefore, I directly cloned from the PCR mix into pENTR/D-TOPO and transform TOP10 cells. I have tried different molecular ratio of insert:vector (0,5:1, 1:1, 2:1), but more colonies I get with 1:1. The next day, I analysed the colonies on each plate.
Problem: The insert from the PCR doesn't go into TOPO vector and instead I got colonies inserted with original template vector. How it's possible that after amplification of my fragment and verification of a single band of it on a gel, the template plasmid has remained in the sample and been transformed into TOP10 instead of my fragment?
After that, I gel-purified my PCR fragment using Qiagen column and then run a gel on the purified product to check that it was really there. The concentration of DNA detected after elution was ~15ng/microliter. Then I repeated the same steps cloning my purified vector into the D-TOPO vector. I also did control TOPO cloning reaction using control DNA template from the kit. However, I didn't get any colonies after transformation or I got colonies with a wrong insert. Control reaction also didn't give me good result. Until now, I am trying to repeat control reaction and clone my purified PCR product into TOPO vector.
Any additional feedback would be truly and greatly appreciated.
Many thanks and cheers!
Hello Yulia,
The first thing is, when you did your PCR for amplifying your gene of interest the template was a plasmid. Now when you use this reaction mixture for ligation into topo vector, the original plasmid will also be present. So when you go for transformation their is a possibility your original plasmid can get into the topo competent cell.
Coming to you problem of not getting the transformed colonies i will give you small suggestions which you can try.
1) competent cells are very sensitive to temperature that is why the company would tell you to store at -20 C. Because when you reduce the temperature it will lose it competence. So first thing is whether your cells are in good condition. If not get new and store it with care. Are you can make them competent by getting protocol from online.(since you mentioned your controls are not getting transformed)
2) when you r doing PCR increase the final extention to 15 min and use it instantly to ligation process. Do not keep it for overnight or store for long time.
If you have any query u can let me know. Good luck.
Hi Nithin,
Many thanks for your answer!
Regarding the competent TOP10 cells I am using for transformation - they provided by the company together with the TOPO kit and I store them at -80C as recommended. I also have my home-lab prepared competent cells which I may try.
Increasing the final extension time can help as i used only 3 min final extension in my protocol and it worked perfectly to amplify a single band of my DNA fragment.
As i red the manual i found that in this technique PCR products are directionally cloned by adding four bases to the forward primer (CACC). so have you added this overhang to the forward primer?? without it this template will not get into the plasmid... and also increase your final extension to 10 minutes.. it may be possible that your enzyme is not replicating the full length template and also making some small frangments, which is unseen on gel because your concentration is low.. but the fragments are there.. and the smaller the fragment its chance to get into the vector is more than the bigger fragment. so even if you are getting single band you should gel purify your band than use it.
Thank you for the amswer, Ramgopal! Yes, my designed primers contain overhangs to make my insert get into the plasmid. The trouble can be in the final extension time that is not enough to amplify the whole length of the fragment. So i will try to play with it.
Yes, to increase the final extension to 10 min is a good point to make sure that all PCR products have the desired length. But I would also recommend to check the manufacturers advice for your polymerase because it could be helpful to increase the extension time in general. For example for Phusion High Fidelity Polymerase you should use 15-30 seconds extension time per kb DNA (2:15 min in your case to be sure) throughout the PCR plus 5-10 minutes final extension. I think that should be the best way to enable the polymerase to amplify the full-length template.
Thanks, Yvonne for your suggestion. I have already did it for the extension time - 2:12min.
Two issues here, 1. first is that you are using a high fidelity polymerase that do not add the A tail that you need for the Topo cloning,.... so adding normal taq maybe a small amount of dntp and 10minutes cycle at 70degrees will do the work, the second issue is that the size of the DNA will vary the ratio, the must be at least 3-1 http://www.promega.com/a/apps/biomath/?calc=ratio
@Alejandro: in contrast to TOPO TA cloning, directional TOPO cloning needs the above mentioned CACC overhangs instead of A overhang (https://www.thermofisher.com/us/en/home/life-science/cloning/topo/topo-resources/directional-topo-cloning.html)
Hi Alejandro. Thanks for your input. Though, as Yvone said above directional TOPO cloning needs particularly CACC overhangs and therefore, Phusion High-Fidelity DNA polymerase is the best option for that. As for the size of my insert, it's almost twice bigger than my vector that can make some complexity during cloning. I am trying to implement 1:1 ratio as it is recommended ifor the insert larger than 3kb.
Hello! To avoid contamination by your original template plasmid why not use restriction enzymes and play directly on the released sequence of interest? Or even take this fragment and clone it in the pENTR/D-TOPO without going through a PCR process if compatible restriction sites exists between the two plasmids?
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