I have ligated 1.3kb fragments to binary vector but it is not showing any single colony after transformation. What could be the problem?
may be your problem lies in digestion, if you have digested PCR product and it is a double digest, it might have happened that your binary vector got digested but PCR product/insert did not, so no self ligants or anything.
Should I check once again that digestion or any other solution to find that?
@ Mariyappan.......
1. Set up a PCR using vector specific primers or one vector specific primer and one gene specific primer to confirm the presence of insert in your vector.....Use 20-50ng of ligated product. If you get amplification, your ligation is fine.
2. Check the efficiency of your competent cells using a positive control. If it is fine. Your cells are working well if not prepare fresh cells.
Also consider point raised by Sadhukhan....
Dear Mariyappan,
the problem could have arisen at multiple stages.
First check your transformation cells with positive control (without doing any restriction on the plasmid of your binary vector) ,most of the time the binary vector wont be suited for the bacteria
secondly- After restricting your binary plasmid with suitable restriction enzymes treat them with alkaline phosphatase , which will prevent your binary vector from self ligating (It should be noted that you should not do alkaline phospahatse treatment to your pcr prdoduct)
If you are cloning a PCR product directly into the binary vector, make sure the primers are phosphorylated otherwise you won't get any clones. You can also treat the PCR products with polynucleotide kinase to add phosphates to the 5' ends
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