I am having problems with cloning 2100 bp fragment which contains selectable marker gene bar (herbicide resistant) with lox site flanking the bar fragment into a vector nearly 13kb. I want to clone the fragment both in Forward and Rev orientation. The recipient vector is a binary vector (pPIPRA560 which already contains Cry1b/1c cassette, Bt gene). I digested both vector in 20 µl reaction with SacII and incubated at 37oC for 4 hours and then inactivated the restriction enzyme at 65oC 20 minutes, and then dephosphorylated the digested vector with 1 µl TSAP (promega) at 37oC for 30 minutes and then deactivated the TSAP at 74 oC for 15 minutes.
At the same time, I have also digested the my insert with SacII in 50 µl reaction using 15 µl DNA and 3 µl enzyme and isolated the fragment using Promega gel extraction kit. Finally, I set up the ligation using standard 1:3 ratio at 16oC for overnight but got no colonies in the plate. I have also tried to set up the ligation at room temperature but the result was same, i.e no colonies. In some cases, I have found some colonies but when I digested the DNA from growing culture of these colonies, I have got band which neither like vector size nor the insert size. I am confident that I have used the right antibiotic in the plate. I am not sure from where this strange object came.
However, can anyone give me suggestion how can I proceed my cloning?
Can you elaborate more about your question?
I am a bit confused when you mentioned "I digested both vectors......." and "At the same time, I have also digested the my insert with SacII in 50 µl......"
My understanding is you are trying to remove an insert a fragment from a vector and then put it into an expression vector (pPIPRA560). Is it correct?
I assume that your insert has both ends compatible with SacII therefore you only use one RE which facilitates flexible orientations (Forward or reverse).
I have some suggestions,
a. Try to increase vector : insert ratio to 1 : 5
b. Try to use several temperatures for incubation (4-22oC)
c. Check your ligase ability by doing re‑circularization
I hope I don't confuse you
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