Assume that the cloned fragment DNA and plasmid have the same cuts by identical restriction enzyme, for instance, Bam HI, is it correct that the foreign DNA could be inserted in a fixed direction? if not, how to ensure the insertion of DNA in such a fixed direction?
Hello,
use one of your vector primers and one of insert primers in a PCR reaction, for example use the forward primer of your plasmid and the reverse primer of your amplified insert to check . you will get bands just if the orientation is correct . Sequencing is also a good option to check the sequence and direction of your clone.
Good luck
You can check the orientation by doing a PCR with one primer belonging to the plasmid and the other to the cloned DNA fragment!
Hello,
use one of your vector primers and one of insert primers in a PCR reaction, for example use the forward primer of your plasmid and the reverse primer of your amplified insert to check . you will get bands just if the orientation is correct . Sequencing is also a good option to check the sequence and direction of your clone.
Good luck
I think you should consider sequencing the plasmid with forward primer and then use the reverse primer for your insert,
If you only do a single restriction digest to end up with two of the same restriction overlap sites on either side of your insert and backbone, you will get your DNA fragment inserted into your backbone in both orientations randomly. You will need to perform strategic PCR amplification and/or DNA sequencing in order to confirm which specific clones are oriented in the way that you want. If you want to avoid this, you can add another restriction site to one of the ends of each DNA fragment using a primer. But you should still sequence your construct to confirm it is what you wanted before using it in any downstream applications.
I agree with Jenifer on this. It is not possible to ensure fixed direction of ligation when using only one RE for digestion as sticky ends at both sides of the insert as well as the vector would be identical and both orientations (forward and reverse) would be equally likely. As mentioned earlier, you could very well go for screening of multiple colonies to select for ones that have the correct orientation but that seems like an awful lot of trouble if your subsequent work does not require sequencing. Directional cloning using two different REs would be much easier. You could try following Molecular Cloning book by Sambrook and Green if you need any help with directional cloning.
Dear friends thank you so much for you suggestions its really helpful for me.
The simplest and fastest way to ensure if the insert is right-oriented is by PCR, using one primer on the insert and the other one on the vector.
Few years ago I cloned a promoter sequence in a vector upstream of the GUS gene using only BamHI and I screened my colonies by PCR (Fw primer on the promoter, Rv primer on the GUS). I got two positive colonies among eight at the first try!
Good luck!
M
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