I've been encountering a really weird problem with cloning. With the same restriction sites, same vector, same procedures, the PCR fragments that's 400bp and 900bp worked fine; however, the 1.1kb and 1.7kb didn't work. I used KpnI and BamHI, which should be easy enzymes. Another thing is, for the two longer fragments, they have quite a bit of colonies on the plate(even more than the other shorter ones), however, they are all just empty... Does anyone have any ideas?
Hi,
The cloning of longer fragments should be less efficient. However, the total size of your desired insert is still small. Is the concentration and quality the same for all four fragments? You need a certain ratio of insert to backbone in your ligation. Since the size of your 1.1 kb insert is almost three times the size of your 400bp insert, you need a three times larger amount of DNA to have the same number of molecules. This ratio could be one explanation for your observation.
Good luck with your cloning!
Hi,
One thing to look at is the buffer for digesting the enzymes. Did you add both enzymes and digested with cutsmart or Buffer 4(NEB)? Try single digest with KpnI first,clean up and then digest with BamHI. Also, as Boas Pucker mentioned check your vector:insert ratio. I often use 1:4. I hope this helps. Good luck with the cloning.
hi...cloning of large fragment is difficult than smaller fragment. You should try two step pcr...
Hi Lan,
First, sounds like you are not efficiently cutting your backbone with your two chosen enzymes, which explains why you are getting many backbone re-ligation colonies. It's good practice to setup a ligation control of your cut and dephosphorylated backbone without insert, and transforming that into E.coli alongside your test ligations to see the proportion of colonies arising from backbone religation in comparison to your test ligations. You don't want to screen hundreds of colonies for the 1 colony that has your colony that has your desired plasmid.
Second, if you are not using the HF (high-fidelity) versions of your enzymes, then you will notice that KpnI and BamHI have some different optimal buffer conditions. NEB offer HF versions of both KpnI and BamHI which have 100% activity in Cutsmart buffer. I'd recommend sticking with HF versions of enzymes as they are all 100% active in Cutsmart. Saves losing material doing sequential digests, purification etc.
Third, the molar quantities of your insert required will change depending on it's size. What I usually do is setup a range of insert:backbone ratios (usually 3:1, 6:1 and 10:1), and run those alongside the cut backbone.
Hope this helps! Cloning can be a pain!
Fragments may be toxic for cells: positive colonies grow slower and may not be seen after o/n culture. Plasmid could be chopped up by the bacteria/DNA rearranged
1. try different strains of bacteria
2. Allow ligation plates to grow for much longer and pick the smaller colonies. Positive colonies grow slow and take much much longer to appear. This worked for us.
3. Check for tandem repeats: Use SURE cells if this is a problem
Hi I am assuming that you had made sure that the PCR fragments are being restriction digested. The other thing is I usually use Recombineering /Gap Repair for larger fragments, it usually very effective. By passes the need of RE and Ligation. While ligating larger fragments dont use Rapid ligation, ligate at 16c O/N. some times adding more DTT and ATP actually helps. As others suggested make sure you try different ratios of Vector to Insert. Also do sequential digestion of your vector to make sure efficient digestion. I usually gel purify at each step. Its a bit tedious but makes your cloning efficient and you can spend less time screening for positives.
it depends on what polymerase you used, the normal taq polymerase? Or HIFI one, for these polymerases which will have different demands. For cloning, I will recommend Gibson aseembly, topo cloning or Gateway those methods rather the T4 ligation.
In addition to all the good advice you've received so far, larger fragments are more sensitive to damage. Minimize or eliminate exposure to light (if you're using EtBr) and use a low temperature to melt your agarose if you're using a gel-purification kit. Alternatively, there are TA cloning kits designed for cloning longer DNA fragments - they take these suggestions into account. Good luck!
If the above suggestion doesn't work. You can try to use high concentration ligase. NEB Ligase is good. U can try ligate at room temperature for few hour and put additional ligase again and incubate at 4C overnight.
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