Hi Guys,
I am inserting a DNA fragment encoding a short peptide into the plasmid. I purchased the fragment with XhoI restriction site at start and the end. The restriction site of XhoI on plasmid will be cleaved. If I insert the fragment in this manner, I will have the fragment in two orientations. Could anyone tell me how to enrich or separate an orientation I want?
Thank You!
I don't think there is a physical way to do it. If you have some way of detecting the expression of the peptide (immunoblotting, for example), then you can transform E. coli cells with the plasmid, choose several colonies, test each one for the peptide, and use one of the clones that express it.
Dear Liu,
I agree with Dr. Adam, its not possible to separate physical both oriented recombinant Plasmids as of same size. One of easy way to transfer the ligated production in to E.coli, and screen the clones with colony PCR by using one vector specific primer and other plasmid backbone specific primer, provided they amplify different size of fragments. All the best,
Dear Dr. Liu,
I agree with Dr. Kuldeep Patel and Dr. Adam Shapiro, once you do the ligation, just by random chance 50% of the resulting constructs that contain you inserted fragment will be in the orientation you want, while the other 50% will be in the opposite direction. The good news is if your ligation is successful, 50% is a good number to work with. You should transform E. coli with your ligation product along with a control, make sure you get more colonies with the ligation than control, and then screen the colonies in one of two ways:
1- Colony PCR as described by Dr. Patel (easiest and fastest way)
2- Purify plasmid DNA (mini preps) from 6-10 colonies and run a diagnostic restriction enzyme digest
Good luck
Wail
I agree with the above suggestions as E. coli will only pick up one plasmid from a mixture (colony PCR or diagnostic digestion) with the clarification on Wail's suggestion: I believe the diagnostic digest he is suggesting would involve restriction enzymes other than the Xho I that you used for cloning.
You would design your diagnostic digest using a restriction site cutting once in the vector backbone and also once within your insert (but not directly in the center of you insert). In this way the size of the excised piece on a gel would identify which orientation your insert lies within the plasmid. You can use a single enzyme, or you may do a double digest depending on what sites are available.
For me, this has at times been very useful for screening, but in the end I always verify sequence in case of errors introduced by PCR (if my fragments were PCR generated). So sometimes it would be unnecessary when I could just sequence 3 or 4 minipreps (if I expect 50% of colonies are what I want) and get both orientation and sequence confirmation all at once. But for you, you probably should not have to worry about sequence errors.
I agree with all the discussed answers. Just want to add few lines.
If you want to confirm the orientation of your gene then you can carry out restriction digestion analysis, using different restriction enzymes. Include also the sites which are present in the gene. And if the orientation is correct you can use that particular clone.
I hope it helps.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology