Ligating blunt adapter to PCR product - (Aug/29/2006 )
Hi All,
Is it possible to ligate the blunt end of a EcoRI-NotI adapter (sticky at the EcoRI end) to a PCR product? I'm concerned about degradation of the AATT EcoRI overhang during the steps before I finally clone into my plasmid. I'd like to ligate the sticky end of the adapter directly to the plasmid, but there is a 2nd NotI site in the plasmid - I've spoken to a post-doc in the lab and he doesn't recommend partial digestion.
i.e. 5'-AATTCGCGGCCGCT-3' blunt-end ligation to PCR product here
............3'-GCGCCGGCGAp-5'
I can't add EcoRI sites to my primers because there are also 2 other EcoRI sites in the PCR product. I have to clone into the EcoRI site - unfortunately, there are no other options. I think I've thought of everything, this problem is now diving me crazy!
Any thoughts?
Edit: Perhaps it is possible to make primers with NotI site, then cut PCR product and adapter with NotI and ligate?
Cheers,
Dave
Nobody?
Hi there,
I do not quite understand the problem that needs to addressed.
But to answer your question. Yes, you can ligate a linker which has an AATT overhang on one end and a blunt end on the other with a vector that has one blunt end and an the appropriate sticky end on the other.
This ligation is not easy in a sense but you can flood the ligation reaction with very high concentration of the adapter (10mM). As long as you flood the ligation reaction, you should not experience problems with degredation of the AATT overhang.
And pardon me for making a stupid comment, but why must you use NotI in the linker if the vector already has its own endorgenous NotI site? How about making a new adaptor? One which can insert into the EcoRI site, killing the EcoRI as it does so and also introducing a new restriction site (say X)which is not found in either plasmid or insert.
Then the insert is amplified with primers that contain restriction site X, thus making an insert flanked by X.
P.S. Partial digest is tricky, doable but tricky. You need colony PCR + multi channel pipette or be willing to do many minipreps (72 or so).
Hi perneseblue,
Thanks so much for taking the time to reply and give some tips.
About your question - well, to be honest, I'm a complete noob in this cloning game, I've been asking more experienced people in my lab lots and lots of questions (which I think they're getting pretty sick of by now!). I didn't even think about making a new adapter. How would I go about doing that? Simply order 2 oligos with complementary basepairing except for the overhang?
Thanks again,
Dave
Thanks so much for taking the time to reply and give some tips.
About your question - well, to be honest, I'm a complete noob in this cloning game, I've been asking more experienced people in my lab lots and lots of questions (which I think they're getting pretty sick of by now!). I didn't even think about making a new adapter. How would I go about doing that? Simply order 2 oligos with complementary basepairing except for the overhang?
Thanks again,
Dave
Absolutely.
The alternative route, and one that might allow you to move your insert to other vectors, is to try to get two enzymes that are unique within your sequence, and unique in the vector (why do you have to use NotI and EcoRI?). Most multiple cloning sites have a range of enzyme sites, and there are a squillion different vectors out there. Heck, even if you only get one enzyme that's unique and useful you will have a 50:50 chance of getting it in the right direction!
Hopefully that will clear things up.
The restriction site carried by the linker must be unique, in that it is not present in either the insert or the vector. The linker may also carry more then one restriction site. With two unique restriction sites, it is possible to make directional ligation of the insert. But in this case, the linker is no longer palidromic. So two oligo need to be purchased.
Try looking at all the restriction sites present. As Swanny has stated there are lots of different Restriction Enzymes. AscI for instance... nice 8bp cutter, should be quite rare.
Try looking at all the restriction sites present. As Swanny has stated there are lots of different Restriction Enzymes. AscI for instance... nice 8bp cutter, should be quite rare.
Hi perneseblue,
I think this strategy is really interesting for a project that I've been trying to get to work, and I'd love to have a bit more information.
Why is it so important to have a unique restriction site in the oligo? If you wanted to cut out a fragment using only one RE, wouldn't it be good to introduce another site of the same type?
How much oligo's would you have to use? I guess it's not too difficult to test out optimal vector:oligo ratios, but a pointer in the right direction would be nice.
I've tried all kinds of stuff to get it to work (most of it including blunt-end ligation) and just haven't been able to make it work. Any help would be appreciated!
Cheers,
Axel
There are many ways to skin a cat, so there are many way to make a plasmid.
Lets consider this very simple situation. Here an DNA insert has to be ligated into a vector at the RE site indicated by the red arrow.
In situation A, four common solution are available.
1- change the vector RE site so that it identical to the RE sites that were used to cut it out.
2- amplify the insert and add new RE site which form compatible ends to the RE in the vector. In this situation a compatible end may mean the use of the same RE (if possible) or the use of an RE which forms a compatible end.
3- A variation of the same idea as Solution 2. Instead the RE site in the vector is changed to become compatible to the insert. (See the similarity to solution 1)
4- Change both inserts and vector, so that a unique RE site is used for the ligation.
Compatible groups can be found on the NEB website. Examples of a compatibility group is the BclI/BglII/BamHI group, NheI/XbaI/SpeI and the SalI/XhoI group. Partial fills can also make compatibility groups, XmaI p.fil/Not p.fil.
In situation B, solution 1 is not possible. As the vector has the same RE site used to excise the insert.
Solutions 2, 3 and 4 remain available. However it is sometimes not possible to use an enzyme which is a member of a compatibility group. So one is stuck with solution 4.
As for your other question, if I had to make a guess, the ideal concentration of linker:vector ratio would most likely be infinity:1
Ligating sticky end linker fragments can be quite difficult. One has to use the highest possible concentration of linker to get it in, as it is easier for the vector to self ligate. I tend to use a linker:vector ratio of 10,000:1 (in mols)
I have never attempted blunt end linker ligation (And never will). Blunt end insert ligation is bad enough.
However, if advice is desired, well
-assuming that the linker only introduces a single type of RE site
- it would be possible to flood the reaction with a concentrated solution of linker. Thus improving chances of vector picking up insert.
- it would help if the linker were 5'phoporylated either by the production company or by addition of PNK.
- it would also help if the ligation reaction was conducted overnight (16hr+) at 4 Celcius.
- alternatively addition of PEG6000 to a concentration of 5% (the same as those quick ligation kits) and incubate at higher temperature like 16 Celcius
- using new ligase buffer and ligase
- dephosporylating the vector (only if the insert is phosphorylated)
- considering a ligation strategy that did not required a blunt end linker ligation!