I read quite a few procedures for the ligation process but I am confused which one is the best or which procedure gives the best ligated result?
Hiii...
Ligation process is dependent on the amount of your vector and your insert along with the temperature at which it is to be carried out. Most of the companies have ligases and HF-ligases that ligates in 10 min at room temperature. You can also do it over-night at 16 degrees C or at 4 degrees C. Mixing of vector and insert is done in molar ratio-3:1, 4:1 or 5:1. You can easily calculate the amount of both via this ratio. There are different websites that can do it for you. You just need to put the length of your insert, vector and amount of vector.
All the best..
Respected Rakesh, i would like to ligate my metagenomic DNA sample into vector i.e. pUC 19 ...... so i would like to know the most reliable or acceptable process of ligation which can give me more positive clones... since i am not able to get more colonies after i do transformation of clones with respect to the control one tht is vector without ligation.......
Respected Piyush, i would just like to clear one thing about the ration you have mention in your answer.... i would like to know ration 3:1 means 3 volume or concentration of Vector or DNA of insert?
Hey.........its molar ratio......
3:1 corresponds to insert :vector. People also call it 1:3 to make it simple as vector:insert.
use this link, if you want....
http://www.insilico.uni-duesseldorf.de/Lig_Input.html
...
for increasing number of colonies you may use electroporation instead of chemical transformation.
Hallo Kashif,
In order to achieve optimal results, you should consider the following:
-The DNA to be ligated should be purified via hi pure purification methods or kits.
- resuspension of DNA should be done in water or in buffer without EDTA.
- try to use your ligation reaction without inactivation( normally heat inactivation drasticaly decreases the transformed colonies (factor >20).
- molar ration of vector: DNA should be 1:3 for sticky ends and for blunt ends 1:5 is recommended.
all best.
Four things:
1. I've never seen the decrease of transformed colonies with heat inactivation and perform it always - and I clone extensively, hundreds of constructs over the last few years. (Actually I learned the opposite thing hehe)
2. There are different condition optimal depending if you do blunt end or sticky end concerning the buffer. Google for PEG-ligation buffers for blunt ended ones.
3. I always add 1mM ATP final freshly to my ligation as the dNTP degrades rather quickly with thaw/freeze cycles of the ligation buffer
4. I use standard now a cycling ligation (8 hours of 30 seconds 10 C and 30 C in thermo cycler alternating, 10 minutes 70 C for heat inactivation). That brings up the background too but it helps tremendously with difficult inserts (introns, viral proteins with strong GC jumps, structured sequences like pre-miRNA).
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