I have been trying to clone a 6.2Kb gene in CMV vector, but i am stuck at the initial stage itself. for PCR i have used LA Taq DNA polymerase/ Accu Taq LA DNA polymerase which produce intense bands at correct size. After gel elusion, i proceed with DNA ethanol precipitation and elute the whole in 4uL MQ.
Using the same i perform Topo Cloning as mentioned in their protocol.
Colonies appear 3-4 on Kana plates, but on miniprep and RD they lack my gene.
Also, i have tried direct digestion of my gene insert and vector with R.E producing staggered ends, But ligation of same produce empty vectors.
I havetried 1:1, 1:3,1:6 vector to insert ratio.
I am unable to understand the reason. After insertion the plasmid size sould be 14Kb, is it not feasible to be cloned in DH5a cells? Or the insert is to big to be cloned? I have heard that Topo Clonin is the best method, but even that doesn't work in my case.
Please help
Hi Debopriya,
Three ideas came to my mind: The transformation efficiency, the TOPO cloning method and the PCR product concentration could explain your results.
1) The transformation efficiency of a 14kb plasmid is usually low. This could be one explanation the low number of colonies you observed. Are you using chemically competent or electrocompetent cells? The transformation efficiency of chemically competent cells might be too low. There are special protocols for the production of competent cells, which can take up very large constructs. However, the use of electrocompetent cells would be easier. Are the plasmids in your colonies empty or is there something else inserted? And is their size as expected? The resulting plasmid of your TOPO cloning should be much smaller than 14kb. I would expect a plasmid size of about 8 or 9kb. It is much easier to transform a plasmid < 10kb than your desired final construct.
2) There are different types of TOPO cloning e.g. blunt and TA. Which method have you used? Only the TA cloning would work, because your Taq polymerase produced PCR products with A overhangs. Nevertheless, you should think about using another polymerase. Proof reading activity could be a big advantage for a 6.1kb fragment! I recommend the Q5 (NEB). It produces blunt end products, which could be cloned via the blunt end TOPO kit.
3) I assume that you got only one clean product in your PCR. In this case, you could try to use the PCR product without DNA extraction from an agarose gel. A simple purification of the PCR product should be enough if this is required by the protocol. The concentration of your 6.1kb fragment is much higher without gel extraction. This could increase the construction of desired plasmids in the following cloning step.
Best,
Boas
Dear Boas
Thank you so much for your advice. I usually go for Topo TA cloning, after ligtion with my insert the size should be 10.1 kb.
I'll follow your suggestions. Thank you so much for your time.
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