Ask a question of molecular biology, fragment connection

1. fragment is a bit long, which makes the connection more difficult. It is suggested to control the molar ratio of vector to fragment 1: 3 ~ 1: 10, and the amount of vector should be as small as possible.

2. If your fragment is a PCR product used for ligation after double enzyme digestion, it is suggested that the enzyme digestion time be longer to ensure that it can be completely cut off, that is, you have to ensure that there is no problem in the ligation process.

If a competent strain fails, I suggest you try other strains.

4. Your fragment and vector size is about 10k. Chemical transformation method is inefficient for large plasmid transformation. You can try electric reform. Good luck!

Do you do double enzyme digestion or single enzyme digestion? Generally speaking, if the target fragment is inserted by single enzyme digestion, the vector needs to be dephosphorylated to avoid self-connection. I'm now constructing a vector linked by single enzyme digestion, so I think there are some things you can discuss. I think the connection is that the dephosphorylation of CIAP can't be completely reacted, which means that some vectors can't be dephosphorylated. Theoretically, there should be some self-connected products in the transformation after connection. You've done it many times, and none of them have grown. Moreover, your competent transformation efficiency is not low, so you can consider changing the ligase, and the ratio of vector to target fragment can be lower when connecting, so as to minimize self-linking. The connection temperature can be 16 degrees or 22 degrees, and it doesn't matter if the time is longer. The ice bath time in the conversion process is not limited to 30 minutes, but can be longer.

I also built some vectors. Generally speaking, I think double enzyme digestion is better than single enzyme digestion. Single enzyme digestion not only increases the burden of screening, but also may lead to reverse insertion. It is suggested to insert the target fragment by double enzyme digestion.

Wish you success!

Thanks for the advice upstairs. When I made the vector, I used a single enzyme for digestion. I made another one yesterday. The enzyme digestion was incomplete this time, so I recycled it directly. Bacteria grew on the plate today, and the amount is not small, but I think it is probably not as long as the cut plasmid. Why can 6.8k be long and 10k not?

Because the concentration of the carrier I made has not been large, I dare not use too little, 3 microliters and 5 microliters, and use rTaq.

I was wrong. T4 Lig is used for enzymes.

The transformation of that ligation product is at least two order of magnitude lower than that of the plasmid,

When you transform, plasmid control is much longer, not just ligation.

When connecting, the amount of vectors and fragments must be thick to connect.