Hi all,
I have tried to clone a 500 bp sh-insert into a pSMD2 vector (4830 bp) for so long, but every time the mini-preps check is not successful. Digestions give the right vector band but never the right insert; its expected lenght after XmaI/SmaI digestion is around 1400 bp, but I always obtain a 900-1000 bp band. I tried to increase the amount of insert for the ligation, but it was completely useless. I need this plasmid to be ready asap, and I am fed up of this cronically unsuccessful cloning. Every suggestion is welcome. Thank you!
I did not get your question.
The size of your insert is 500 bp and the size of your pSMD2 vector is 4830 bp. Then 1400 bp is expected to be the size of what fragment? I did not get this point. What is your problem? Would you clarify your question?
the problem is that I never get the expected 1400 bp after a specific enzymatic digestion (I perform a double-check of my colonies, by using a combination of two restriction enzymes for ONE digestion, and only ONE enzyme for the other digestion). Is it clearer now?
Can you do a colony PCR to check if the fragment was inserted? In this case you don't need to do a restriction digest.
Sometimes the problem is in the T4 ligase, I suggest you to do the ligation over night at 16C and if it doesn't work probably it is because the T4 ligase is too old.
Good luck
Thank you Marta.
Actually I figured out that all the enzymes were too old, so I restarted the cloning by using brand new enzymes and buffers. A colleague of mine had the same problem with another cloning protocol, but it was solved by using new enzymes.
Thanks a lot.
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