will large size 1,8kb of PCR affects ligation with 5.3kb of plasmid? I have repeated this three times; still, I'm not getting it, why it is not happening?
Hi Praveen
Did you digest both the insert as well as the plasmid before proceeding to ligation?
Assuming that the ends are compatible, the DNA sizes should not be the problem. If the DNA is blunt ended, please try longer incubation time.
Also, please share the following information:
1. Vector and Insert DNA A260/280 and A260/230 ratios before adding to ligation mix. Contaminants or phenol carryover from miniprep can interfere in ligation. Did you dilute your DNA before adding to the reaction mix? Dilution can help reduce interference from contamination.
2. Ligation reaction temperature and duration. Sometimes, a reaction does not work in 2 hours at 16degreeC. In such cases, leave it overnight in a sealed ice box. As the ice melts, the optimum temperature is achieved at some point and ligation occurs.
3. Ligation reaction volume: This should be not more than 20uL. Larger volumes decrease the chances of successful ligation.
All the best ahead for the ligation :)
If you are trying to ligate the PCR product blunt with no digest step, make sure you are using a polymerase that doesn't leave an over-hangiing base at the end. Phusion is a good one to choose. The low-fidelity Taq polymerases leave a 3' A.
If it is indeed a blunt ligation, you will have to use a kinase to phosphorylate the ends of the PCR product, and a phosphatase on the cut vector to avoid ligating the empty vector shut again.
If you are using T4 ligase, 16C is the optimum temperature for an overnight reaction.
I also cannot stress enough the correct handling of ligase buffer - it contains both ATP and DTT. The DTT is not stable at room temperature for long, so always thaw the buffer on ice. The DTT also precipitates out (often visible as small flecks), so before you add it to your reaction you should vortex it, and then vortex it again to make sure. When I receive a new tube of buffer I aliquot it into 20ul lots and only thaw an aliquot once, vortexing before use.
If you suspect the DTT has gone off then you can spike it with some fresh stuff, but always best to use a fresh tube of buffer. Kits usually come with two tubes for this reason.
I hope this helps. If you are not doing a blunt reaction, could you please give more details of your cloning design?
Hi,
I totally agree with Malini. I just want to point out other considerations you should also take an account.
I don't know you calculated or not but, you have to calculate your vector:insert ratio at first. In general, vector :insert ratio is used 1:1 or 1:3. You have to optimize your ratio to test different vector:insert ratios (1:1, 1:2, 1:3, 1:5). There are very useful online tools you can use. I just gave one below.
http://nebiocalculator.neb.com/#!/ligation
The other thing is the conditions of the ligation enzyme you use must be checked. 16, 25 or 37 C degrees, ATP concentrations etc.
Please check it is expire date of the ligase too.
Good luck
use two different enzymes that produces overhang at two different ends. use 5 to 10 times more insert than vector. try overnight ligation with T4 ligase. these may help.
Hi Praveen
The insert is a PCR amplified product. Is the cleavage close to the ends of the fragment? If yes, did you provide enough extra bases at the 5' ends of the primers for restriction enzymes to attach?
Please see this
https://www.neb.com/tools-and-resources/usage-guidelines/cleavage-close-to-the-end-of-dna-fragments
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