Hi everyone, my labmate and I have been trying to clone a few shRNA constructs in the past 2 months. We encountered quite a few issues and only successfully cloned 2 out of 18 constructs so far. I would like to ask for some advice from you. Thank you so much in advanced! Below are my questions and link to brief description of our protocol + result. Please let me know if there is anything else you'd like to know.
Best,
Huong
- Our high background indicated that the CIP didn't work very reliably and that the vectors were not fully digested by both enzymes. How to fix this? Maybe to use other phosphatase? serially digest the vectors?
- Do you have recommendations on how to make sure CIP works properly? If not CIP, how to efficiently get rid of/reduce the background?
- What is your protocol for annealing the oligos (temperature setting, buffer, concentration to start with, etc)?
- How effective is the phosphorylation with T4 PNK? How to tell if the reactions actually work? Maybe by running gel?
- Did you follow NEB recommendation for total DNA concentration during ligation? If not, what did you usually use? And what is the optimal ratio of vector:insert?
- Out of the 9 positive clones that we got, 3 of them have some sort of mutations. Could the bacteria be accounted for this? If so, which other competent cells are better?
- Our successful constructs are for the same domains on two proteins. Could there be a favor toward these oligos? How to enhance the success rate of the others?
Link to protocol and result is attached! Thank you all!
https://www.evernote.com/l/ANrmDsWxpTZPQbL7c4c_tE2R1w9_4aKmK7Q
Hello Huong Ha
Here's my suggestion.
- Our high background indicated that the CIP didn't work very reliably and that the vectors were not fully digested by both enzymes. How to fix this? Maybe to use other phosphatase? serially digest the vectors?
==> Why don’t you try to do single digestion (either BgIII or HindIII)?
Then, check your linear band. If you see one linear band, you can try to do another digestion. Sometimes double digestion is not perfectly working. I think this step is very critical.Then, the rest of procedures (CIP treatment or annealing or T4 treatment etc.,) will be working.
- Did you follow NEB recommendation for total DNA concentration during ligation? If not, what did you usually use? And what is the optimal ratio of vector:insert?
==> I used 1:3 , 1:6 , 1: 10 (v : I) . These ratio of V:I will be working. When you do transformation, you also need to add three negative control groups (insert alone , linear vector alone, and no vector ).
Good luck !
Chung Sub
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