Hi all,
Hope everything good. I came across a problem.
My NEB enzyme xho1 and Nhe1HF did not work. I check them one by one using PCAGGS, and it looks good. The xho1 have a little worse efficiency at 40min, but after 90min, they all digest well.
So i use Nhe1HF digest PCAGGS, do gel purification. The concentration is 149ng/ul. And then use xho1 digest the product, 14ul Nhe1HF digest product and 1.5ul xho1. do gel purification. The final concentration is 61ng/ul.
I use the product vector 1ul ligate with my insert by T4 ligation. I also add a control, only vector do T4 ligation.
And do transformation: 2ul ligation product with 60ul xl-blue cell.
But the next day, show a lot colony on the control one. Could you guys give me some suggestions. Thank you very much!
Hi Yijie Wu.
Your enzyme is compatible for digestion together? I think most NEB enzymes are compatible in their supplied buffer. Do you linearize your insert together? Also, did you linearize your vector with the same enzymes?
Have you try running digested control for your vector. Ideally you would expect no colony. If there's a lot of colonies, it means your REs digestion have some issues. It didn't linearize your vector enough for the insert to be cloned into.
Usually I use all 20uL ligated product into 100uL competent cells. I want to increase the chance of the insert and vector to get cloned together and willing to trade off the cell's viability. As long as few cells take up the insert, I could propagate the transformed cells. However, it's up to you on what you're willing to trade/risk off during the transformation.
In good news, your transformation protocol worked great! That means you can rule out issues with cell viability and the transformation process.
Do a double-digest in CutSmart Buffer & heat-inactivate. That way you won't lose so much of the product in clean-up steps. https://nebcloner.neb.com/#!/redigest
Did you digest the vector with the same enzymes? Did you phosphatase-treat the vector?
I'd say chat with the lab manager, a postdoc, or your advisor. They can give other good, practical advice. Good luck!
How close to each other are the sites? If they are very close than it is possible that once one cut accrues the other site is no longer cleavable.
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