In the absence of further details, here are some comments:
1. The undigested control is covalently closed, supercoiled DNA. Accordingly, it migrates faster than the linearized forms, as seen with the vector pLKO.1 puro digested with AgeI, MluI, and AgeI + MluI.
2. It is difficult to interpret the digestion pattern of pLKO.1-shDys. What is the length of the shDys segment ? Was the ligation at the AgeI site supposed to reconstitute the site, or did the cloned fragment just contain a compatible sticky end extension ? Does the shDys gene contain an MluI site, and if so, where ? Why not digest with the two enzymes whose sites are supposed to flank the insert, i.e. AgeI and EcoRI ?
In general, it is far easier and faster to screen clones for the presence of the desired insert by colony PCR with appropriate primers that doing mini- and maxipreps plus restriction digests.
1. What is the length of the shDys segment?
-It is 1759bp, so the inserted vector length is 8784bp.
2. Was the ligation at the AgeI site supposed to reconstitute the site,
-I have no idea..
did the cloned fragment just contain a compatible sticky end extension?
-yes it does.
Does the shDys gene contain an MluI site, and if so, where ?
shDys gene does not contain MIuI site. further answer is on below
Why not digest with the two enzymes whose sites are supposed to flank the insert, i.e. AgeI and EcoRI?
- I was supposed to use the MIuI and EcoRI so that if the target gene is inserted, the MIuI site will be disappeared and the outcome will be similar to the outcomes of AgeI. But i think it is not..
I really thank you for your answer. I will do it again with your reference.
Sincerly, Pierre Béguin .
Please, just ignore my suggestions if your problem is already solved with Pierre's help.
Unfortunately AgeI does not cut after ligation. With a proper design the site should be reconstituted (check with your adviser). The MluI site is destroyed as expected. However the EcoRI/MluI digest of the vector and after ligation appear to result in fragments of similar length which suggest that your fragment is not inserted. You may load these digests side by side to better visualize a size difference or use an enzyme that cuts your vector at multiple sites (e.g. HindIII).
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