Dear Researchers!
I want to make the knock-in vector to construct the knock-in cell line.
But my ligation experiment dose not work (Always backbone vector was self-ligation)
I just let you know that knock-in vector information of each part!
- 5` homologous arm: 550 bp
- 3` homologous arm: 610 bp
- knock-in gene: 3495 bp
- Backbone vector: 3500 bp
When I was trying to ligation each part, use the in-fusion cloning kit.
But it was not working....
How can I make it?
Are you using Takara's infusion cloning kit?? What ratio of insert to backbone DNA you are using? I think that is the most critical part of infusion cloning. We have to take the insert to backbone ratio around 3:1. If you increase the amount of backbone DNA, it will give you least no of vectors having the insert and more no of vectors getting self ligated.
Mohd Ahmad Thank you for your comment. I take the insert to backbone ratio 2:2:2:1 (each insert 2, backbone 1) using Takara's infusion cloning kit. I will try to increase the insert ratio. And I have one more question. My backbone vector size is 3.4 kb and one of insert size is 3.4 kb. If insert size was same or bigger than backbone vector, cloning efficiency will be decresing?
I need to change the backbone vector?
Dear Seok-Hwan Song
From the above information it is not clear that how many insert you are trying to clone into a backbone. If you can explain the following points then it will be easier to help you.
1) How many insert are there, and what is the length of each fragment?
2) What is the length of homologous overlapping region in insert with respect to backbone and vice versa?
It should be minimum 15bp. You can increase it upto 20-30 bp.
3) When PCR amplifying the fragments, did you get the single specific band?
Its always good to run PCR sample in agarose gel, and cut out the specific band only and gel extract this DNA sample. Presence of unspecific bands can lower the efficiency.
4) Have you PCR amplified the backbone or it was digested with restriction enzymes to make linear fragment?
It should always be a PCR amplified product. After extracting the specific band from agarose gel you can try to treat it with Dpn1 restriction enzyme to destroy the circular plasmids in the background. Because sometimes this template can cause vector only background colonies.
5) Have you checked the positive control provided in kit?
It will help you to troubleshoot that your kit reagents are working fine, and the problem lies somewhere else.
6) What incubation time and temperature you are using for this reaction?
Best.
Mohd Ahmad
Thank you for your help!
I was explaining for your questions.
1) Total insert number: 3 ea
insert size: 550, 613 bp (homologous arm), 3.5 kb (gene)
2) each gene has 20 bp of homologous overlapping region.
3) When I run a PCR, specific band was amplified with nonspecific band. After electrophresis, specific band was cut and elution.
4) In order to make linear fragment of Backbone vector, it was cut by restrction enzyme.
5) Not yet. But I made knock-in vector using infusion cloning kit, previously.
6) I have followed manufacture protocol. Mixture of infusion cloning reaction was incubated for 15 min at 37 degree then 15 min at 50 degree and place on ice.
Please check my answer and if you have any question, please let me know.
Thank you very much for help me!
Dear Seok-Hwan Song
Your vector backbone should always be a PCR amplified product. If you treat the plasmid with restriction enzymes to make the linear backbone fragment and use it in infusion reaction, then it will cause the "vector only background colonies" on plate. The reason for this problem is that when digesting the plasmid, not all of the plasmid copies get digested and some remains in the circular intact form. After transformation they replicate and cause the fake colony appearances on LB-agar plate.
I guess this is the reason for vector backbone only colonies.
I would like to suggest you to PCR amplify the backbone fragment then extract the specific band from agarose gel and then use it in infusion reaction.
Best.
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