Hello,
I am extracting mouse DNAs and performing PCRs through KAPA 2G Fast hotstart genotyping kit. Before I use the pcr products for TA Cloning (invitrogen), I am purifying PCR products through gel purification and PCR purification (Qiagen) but the DNA concentrations are too low (around 2-5ng/ul). When I continued for the cloning, I didnt get any colonies(only transparent color colonies were in the plate). Does Kapa genotyping kit not suitable for TA cloning? I couldnt figure it out why the concentrations of DNAs are too low? How can I increase them? Should use different kit for extraction and PCRs or If I use Tag polymerase as suggested in the kit, it would be better? I would be glad if you be able to provide me some input.
Best Regards,
Dilara
Dear Dilara,
From the product datasheet (https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Roche/Datasheet/1/2gfhsgkbdat.pdf) "DNA fragments generated with KAPA2G Fast Genotyping Mix are 3’-dA-tailed and may be cloned into TA cloning vectors". Any product should be apt for TA cloning as long as it has an 3'A overhang, so I suspect you are either recovering non A-tailed product or recovering too little of it.
Assuming you are using a TOPO vector for TA cloning, I would recomend NOT to purify the product by gel but rather clone it immediately. If you have to (like have multiple bands that are more abundant) I would recomend to A-tail again after gel purification by adding dNTPs mixture (or only the dATP), Taq buffer, MgCl2 and Taq Pol, like for a regular PCR, and incubate at 72 for 10 min, then transform competent cells. If you are using electro-compentent cells you'll probably have to dilute not no arc the thing.
Regarding the low recovery of the gel band, there are a few things that work for me (besides avoiding gel recovery altogether when possible) :
1. Use low % agarose gels with TAE buffer (rather than TBE, since borate-agarose is harder to dissolve)
2. Incubate the recommended 10 min at 55C with shaking and make sure there is no undissolved agarose
3. If your band is heavy (like 10Kb, which I assume is not since is form genotyping) add 1 vol of water and 1 of isopropanol to the dissolved agarose before putting through the column (this is a recomendation from zymo's kit)
4. Use Zymo's or Minelute columns, since they allow for elution in very small volume, which will be concentrated.
5. Be careful not to carry any of the agarose dissolving buffer after the first elution (don't let the tip touch the eluted solution).
6. Heat the elution buffer to 60 C and let it stand in the silica column before eluting for 5 min.
Best of luck.
Dear Jose,
Thank you so much for your comment! It helped me a lot!
Best Regards,
Dilara
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