I have tried to clone a specific gene into pCAMBIA1304 vector. But all the attempts failed. I used ligation kit from both Promega Corporation and Takara Bio. However, there was one time I used Takara Bio T4 DNA ligation kit and obtained few colonies on the tranformation. But when i screened using the colony PCR, the size of the amplicon is smaller (approx.300bp) compared to the actual size (approx. 400bp) of the gene of interest. There were no any problem with my positive and negative control. Can anyone suggest me on what I can to to resolve this issue?
Thank you.
Use fresh amplicons or end repair if amplicons are not fresh. Use blue white selection for better selection.
Some kits are better than others and there is no generalization, in my experience. You can just try and find better kit in your case. As you said kit from Takara worked for you, use it with blue white selection for getting good number of positive colonies. Try to follow the protocol and trouble shooting guide.
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