ligation failure-what to do? - tried everything but still not getting transformants (Mar/06/2008 )
i'm trying to ligate a 1 kb insert into my vectors, digested with nco1 and xma1. i ligated the insert-vector using t4 ligase at 16 degrees celcius, overnight. when i did the transformation (heat shock, 1 min at 42 degrees celcius) into e. coli xl1 blue, all i got was two colonies and there were colonies on my control plates too (control plates were just the digested vectors without the insert). my negative plate (competent cells with no ligated product) however was clean. so it's not an antibiotic problem.
what can i do to improve the quality of my ligation? what are the important factors to consider in a ligation procedure?
It sounds like a pretty simple ligation which makes me think something simple is wrong.
Firstly, make sure you haven't made any simple errors such as designing the incorrect restriction site in your primers or using an incompatible buffer in your digests. Your primers should have at least a few flanking bases outside the restriction site as well.
Vector preparation is probably the most important thing in a ligation. The vector needs to be dephosphorylated. Even vectors prepared with two non-compatible restriction enzymes need to be dephosphorylated to prevent the self-ligation of vectors that have only been cut once. The vector stock also needs to be good quality, it should be grown from a single colony. The quality of the vector stock can be determined by digesting it in several locations and checking to make sure only the correct bands appear in the screen.
Your reagents are probably the next important thing. Make sure your reagents are working well. The low numbers of colonies on your plates suggest your transformations may not be working well - measure your transformation efficiency and use a positive control such as the vector stock in your next transformation. Test your restriction enzymes to make sure they are working. You can digest your vector with each restriction enzyme and another restriction enzyme to produce a good sized band to confirm that both your cloning enzymes are working.
Assuming all your reagents are working well, try a ligation with more insert. Also, i usually incubate my ligations for 1 hour at room temperature and transform that. Then i incubate my ligation O/N at 16C and if the first transformation doesn't work, then i transform the 16C ligation.
Good luck,
Rob
are you sure that your ligase is active? try a test ligation and check it on a gel.
In addition to all the above suggestions, several additional suggestions for you:
1. Make sure your vector is completely digested. You may want to digest your vector overnight at 37oC. Also, please do not add in too much vector DNA in your digestion. I found that too much vector DNA always result in incomplete digestion. Ideally, in 50uL reaction, it is preferred to add in 20 - 30 uL of vector DNA for digestion.
2. Since you are doing overnight ligation, why not you set up the ligation at 4oC?
3. You may consider to clone your fragment into a TA cloning vector and digest your insert from the recombinant TA construct, purify the insert and clone into your vector.
4. Never have TE buffer in your DNA sample. H2O is sufficient. EDTA in the TE buffer may interfere with your enzyme activity.
Hope this will help you.
All the best.
1. Make sure your vector is completely digested. You may want to digest your vector overnight at 37oC. Also, please do not add in too much vector DNA in your digestion. I found that too much vector DNA always result in incomplete digestion. Ideally, in 50uL reaction, it is preferred to add in 20 - 30 uL of vector DNA for digestion.
2. Since you are doing overnight ligation, why not you set up the ligation at 4oC?
3. You may consider to clone your fragment into a TA cloning vector and digest your insert from the recombinant TA construct, purify the insert and clone into your vector.
4. Never have TE buffer in your DNA sample. H2O is sufficient. EDTA in the TE buffer may interfere with your enzyme activity.
Hope this will help you.
All the best.
I always use TE buffer to elute my DNA. This is because the pH of our water here is acidic (<5), which I didnt know and had to find out after countless failed purifications, etc. Alternatively, I use 1/10 diluted TE buffer to get the lower EDTA concentration but still get my DNA in a buffer solution at roughly neutral pH.
Ive also had a HUGE number of problems with my ligations for the past 6 months. I havent been able to make a single construct succesfully. Before that everything worked well and im doing things in exactly the same way with new buffers and enzymes and ive tried all the possible tricks in the book. Nothing works. Maybe it has something to do with the weather
1. Make sure your vector is completely digested. You may want to digest your vector overnight at 37oC. Also, please do not add in too much vector DNA in your digestion. I found that too much vector DNA always result in incomplete digestion. Ideally, in 50uL reaction, it is preferred to add in 20 - 30 uL of vector DNA for digestion.
2. Since you are doing overnight ligation, why not you set up the ligation at 4oC?
3. You may consider to clone your fragment into a TA cloning vector and digest your insert from the recombinant TA construct, purify the insert and clone into your vector.
4. Never have TE buffer in your DNA sample. H2O is sufficient. EDTA in the TE buffer may interfere with your enzyme activity.
Hope this will help you.
All the best.
I always use TE buffer to elute my DNA. This is because the pH of our water here is acidic (<5), which I didnt know and had to find out after countless failed purifications, etc. Alternatively, I use 1/10 diluted TE buffer to get the lower EDTA concentration but still get my DNA in a buffer solution at roughly neutral pH.
Ive also had a HUGE number of problems with my ligations for the past 6 months. I havent been able to make a single construct succesfully. Before that everything worked well and im doing things in exactly the same way with new buffers and enzymes and ive tried all the possible tricks in the book. Nothing works. Maybe it has something to do with the weather
Hi ya!
You may also use Tris buffer (Elution buffer in Qiagen plasmid extraction kit) to dissolve your DNA. As long as you get rid of EDTA in your sample. Alternatively, you may also purchase molecular biology grade water from company.
Some restriction enzymes might have problems in cleaving at the end of a PCR product. Do not know if this is the case for your restriction enzymes. But if it is, then it might help to use a blunt end ligation kit (like blunt TOPO-kit or cloneJET-kit). Cutting your fragment from a vector might help cutting efficiency and thereby help ligation into your vector of choice.
I am not sure if you can find this useful piece of information in every catalogue for restrisction enzymes. In Promega catalogue, there is a table listing the number of additional nucleotides that is required before a RE cutting site, in order for the RE to cut the PCR fragment effectively.
By the way, I personally think that cloning a PCR fragment into TA cloning vector and digest the fragment from the construct is the safest way, which guarantees the complete digest of the insert, though it is a little time consuming.
Check your ligase buffer. The buffer contains ATP to drive the reaction and it regularly precipitates out of solution when you freeze it.
Thaw the buffer and look for any signs of precipitation. Vortex until it is all clear.
Many times my students have ligation problems and it is because their buffer was not well-mixed prior to use.
Also, check the design on your insert restriction sites. If you are adding your own restriction sites to the ends of primers, you need to account for a few extra basepairs the enzymes need to work efficiently.
Check out the appendix section of the New England Biolabs catalog for a nice table that lists how many extra nucleotides you need to add.