I want to insert 4 restriction sites between my two genes. Initially, I thought of inserting MCS into my vector and then cloning my two genes on either side of the MCS.
But, is it possible to add 2 or more REs on my primer ends and do PCR?
Will it work?
I want to add XhoI SacII ApaI BglII REs between my genes A and B.
When amplifying gene A, can I add two REs (XhoI SacII) in its reverse primer?
When amplifying gene B, can I add the other three REs (SacII ApaI BglII) in the forward primer?
So that I can cut the SacII site and ligate A and B and finally I will have:
A - XhoI SacII ApaI BglII - B
You can add as many as you like!
Main thing to watch out for is whether the chosen enzyme cuts efficiently when it is near the end of a DNA fragment. There is a very good guide for this:
https://www.neb.com/tools-and-resources/usage-guidelines/cleavage-close-to-the-end-of-dna-fragments
It is always a good idea to have at least 4 bases before the first cut site on the primer.
Dear Vidhya,
Rightly said by Shah that you can add as many as you want. But, you need to design the primers carefully to prevent compromising the efficiency of the restriction enzymes when you might want to use multiple enzymes at a time. If not then go ahead. Otherwise, just take into account the buffer compatibility of the enzymes you want to use together, the flanking regions required with the restriction site. If the sites are too close to each other then binding of one enzyme will exclude the binding of the other. Both the length and the sequence of nucleotides between the restriction sites should be carefully chosen which would favour the digestion.
Hope it helps!
hi
as mentioned above as many RS u want u can add.
just take inti account the enzymes compatibility during restriction digestion.
and use of buffers compatible for both the enzymes and if possible digest with one enzyme at a time.
I wanted to ask a question to Deepan Shah. The NEB site has two different lists which vary on the number of nucleotides required at the flanked regions of restriction site. For example, the new table shows NdeI shows activity indicated by +++ (50-100%) even with only 3 bp flanking region whereas the old one (link attached) shows NdeI needs at least 7-8 bp for activity! With lesser than that even 20 hours of incubation there is 0% activity. So, which one do you think should be followed?
https://www.neb.com/~/media/NebUs/Files/Chart%20image/cleavage_olignucleotides_old.pdf
Response to Tias Saha:
The differences in results reflect the differences in methodology. They have three separate charts:
1. In the chart you attach, they used annealed synthetic oligonucleotides with equal number of bases flanking the restriction site.
2. In the one I attached, they used annealed synthetic oligos with 12 bases on one side of the restriction site and increasing numbers on the other side.
3. A third chart describes the results from linearised vectors and shows some differences from the other two.
Therefore the context of the restriction site is very important. In the case where you have many sites close together, you have to consider this and may need to carry out sequential cuts with one enzyme first and then the second enzyme - being the one which tolerates the edge best.
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