digestion of insert twice and ligation of 3 bits - (Jul/16/2010 )
Hi guys,
I need to generate a construct with 2 inserts and to make it more complicated (for the mol biol novice at least), I need to digest one of the inserts twice.
My questions are:
Is that OK that after the 1st digestion when I get insert#1 with an extra bit, I just digest the fragment for the 2nd time to get rid of the extra bit or do I need to clone it into another vector and do the 2nd digestion on this temporary construct? If the latter, then what vector should I use for that?
Once I have the right fragments, can I just ligate all the 3 bits (2 inserts and empty vector) in one reaction or do I need to do it in two steps? If two steps, then in what order should I do it: 2 inserts ligated together and then with vector or insert+vector then 2nd insert?
Thank you so much for help.
for the insert that you need to digest twice, how many enzymes total do you need to use?
if the answer is 2, you can just double digest it.
if the answer is 3, i've heard a triple digest works provided the buffer works for all 3.
if the answer is 4, you can digest it with the first set of enzymes, gel purify the fragment, then digest again with the second set of enzymes and gel purify again. you could also digest it with the first set of enzymes, pcr prufiy, then digest with the second set of enzymes then gel purify to use for ligations.
the reason i suggested the method of gel purifying twice even though it would be harder than pcr purify then gel purify is because i've had to do this before because the enzymes i originally wanted to use produced fragments of the vector the same size of my insert. so to solve the problem i had to digest my fragment out with two enzymes that woudlnt cut my vector internally, gel purify that fragment, then digest again with my desired enzymes which would have originally generated my insert + another fragment of my vector which was the same size. if that sounds confusing and doesn't relate to you then don't worry about it.
To answer your question of ligations, a ligation of two inserts and one vector works fine. i usually do a 1:3:3 ratio of vector:insert:insert. molar ratios of course.
actually a quadruple digest works too, provided all enzymes can work in the same buffer. Just keep the total volume of all 4 restriction enzymes below 5% of the digest volume.
In multiway ligation, I use a mol ratio of vector to inserts at 1:1:1.
So as you can see, there is some leeway.
perneseblue on Jul 17 2010, 03:35 PM said:
In multiway ligation, I use a mol ratio of vector to inserts at 1:1:1.
So as you can see, there is some leeway.
off topic but do you know if there's any limit to how many enzymes you can digest with at once? provided the buffer works for all and the % glycerol is ok.
Thanks a lot, that is exactly why I need to digest a fragment twice, one of my enzymes would cut the vector as well at similar size.
Thanks for the quick response.
Krisztina
molstudent on Jul 17 2010, 07:29 PM said:
if the answer is 2, you can just double digest it.
if the answer is 3, i've heard a triple digest works provided the buffer works for all 3.
if the answer is 4, you can digest it with the first set of enzymes, gel purify the fragment, then digest again with the second set of enzymes and gel purify again. you could also digest it with the first set of enzymes, pcr prufiy, then digest with the second set of enzymes then gel purify to use for ligations.
the reason i suggested the method of gel purifying twice even though it would be harder than pcr purify then gel purify is because i've had to do this before because the enzymes i originally wanted to use produced fragments of the vector the same size of my insert. so to solve the problem i had to digest my fragment out with two enzymes that woudlnt cut my vector internally, gel purify that fragment, then digest again with my desired enzymes which would have originally generated my insert + another fragment of my vector which was the same size. if that sounds confusing and doesn't relate to you then don't worry about it.
To answer your question of ligations, a ligation of two inserts and one vector works fine. i usually do a 1:3:3 ratio of vector:insert:insert. molar ratios of course.
Thanks very much Perneseblue, I feel a lot more confident now.
perneseblue on Jul 17 2010, 11:35 PM said:
In multiway ligation, I use a mol ratio of vector to inserts at 1:1:1.
So as you can see, there is some leeway.
molstudent on Jul 18 2010, 09:15 PM said:
No, I don't.
Personally I don't think there is a limit provided all enzymes are happy to share the same buffer and the glycerol % is below 5% total volume.