I have tried the ligation procedure quite many times, but have failed to get colonies after transformation. I get no colonies after transformation. Also I have tested the competence of competent cells and they are working fine at 10ng of plasmid concentration. What can be the possible reason for the failure in ligation?
If you suspect that either your T4 DNA ligase or its buffer (ATP in buffer can go bad following repeated freeze thaw or prolonged storage at incorrect temperature) has gone bad, incubate 1-2 uL of HindIII digest of lambda (lambda HindIII ladder) in a total of 10 uL reaction and run the entire reaction on agarose gel. If your ligase and buffer are good, you will see a predominant high molecular weight band otherwise a ladder.
Reasons for failed ligation:
There are lots of things that can go wrong from bad vector and insert DNA to incorrect vector/insert molar ratio. Most ligations fail because DNA fragments purified from gels were exposed to UV for extended periods of time that results in nicking of the DNA. The purified DNA fragments were contaminated with chaotropic salts (used in Gel/PCR purification kits) or Phenol etc.
To clarify Syed's point:
You can test your ligation reactions by running a series of digestion/ligation reactions out on an agarose gel with EtBr or SYBR staining. I’d recommend some or all of these reactions, assuming you have two restriction enzyme cut sites you may want to test.
1) Undigested plasmid
a. Will migrate as an undigested plasmid usually does.
2) Digested plasmid (Enzyme 1)
a. Will migrate as a linear dsDNA, above ‘1’
3) Digested plasmid (Enzyme 2…)
a. Will migrate as a linear dsDNA, above ‘1’ (identical to ‘2’)
4) Digested Plasmid (Enzyme 1+2…)
a. Will migrate as two dsDNAs, allowing you to check cutting efficiency and confirm the expected fragment size.
5) Undigested plasmid plus ligase
a. Should be unaffected by ligation, and look identical to ‘1’
6) Digested Plasmid +ligase (enzyme 1)
a. Will migrate as a ladder of concatemeric plasmids if ligation occurs.
b. If ligation fails, it will look like ‘2’ and ‘3’ (use this to measure efficiency)
7) Digested Plasmid +ligase (enzyme 2…)
a. Will migrate as a ladder of concatemeric plasmids if ligation occurs.
b. If ligation fails, it will look like ‘2’ and ‘3’ (use this to measure efficiency)
8) Digested Plasmid +ligase (enzyme 1+2…)
a. Will migrate as a ladder of concatemeric plasmids and inserts if ligation occurs.
b. If ligation fails, it will look like ‘4’
Following this general protocol you can examine the ligation efficiency at both cut sites. This will burn through a fair amount of plasmid and enzyme, but it will give you an idea of what is going on with your reactions.
It’s up to you whether it’s worth the work.
Failure of a ligation is not the only reason you might end up with no colonies after transformation. Trying to transform too much volume of a ligation reaction or to much DNA into competent cells can result in no colonies as well. A general rule is to transform no more than 1/10th of the volume of competent cells (i.e. 5ul ligation into 50ul cells) and no more than 40ng of DNA. Most of the time 1ul of a ligation transformed into 35-50ul cells is sufficient to produce colonies and obtain your desired clone.
Good Luck!
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