I am currently working with a final destination vector (gateway technology) that I want to replace a gene native promoter and replace it with CaMV35S promoter. I have cut my vector (pKDWG2R) with HindIII(5') and SpeI(3'), and was able to purify my vector (length 12.599 Kbp) obtaining a concentration of 14ng/ul with a ratio (260/280) of 1.87. I synthesized the CaMV35S promoter with KOD polymerase from pEARLYGATE100 final destination vector with HindIII(5') and SpeI (3') restriction sites. I obtained a 346bp product that was purified and digested with the respective enzymes for 2 hours, digested insert has a concentration of 61ng/ul. Then I proceed to ligate my vector with insert (molar ratio 3 insert: 1 vector). I used 100ng and 120ng of vector. I used 1U of T4 DNA ligase (promega) in 10l of reaction. I have incubated at RT (3hours), 4 degree (overnight), and 16 degree (overnight). This binary vector (pKDWG2R) contains Spectinomycin resistance gene (bacteria) and Kanamycin resistance gene (plant). Since this vector contains ccdB gene, I have been transforming my ligation product into db3.1 competent cells. However, i have not seen any colony formation. I am just wondering whether the length of my vector is influencing the reaction, due to when I do my calculation to keep the molar ratio 3:1; i ended up adding 9ng of my insert, which in my opinion is really low.
I would like to receive feedback about whether or not I am performing well the ligation or any advice.
Thanks in advance
Did you had adittional overhangs at your restriction sides for the insert?
A lot of Restriction Enzymes, or I encountered nearly all I used work better, when there are additional nucleotides in both directions of your restriction sides. You can try to subclone your insert into the pJet 1.2 System and then digest it from that again for further cloning.
For Ligation you can try a temperature gradient ligation over night. Just use an ice bad and put your ligation in it. It now goes over night every temperature step and maybe this works. Or you could change your ligation volume. Sometimes it works better in a higher volume we normally use 25 ord 35 µL for ligation.
Hello Edgard,
To troubleshoot, I would recoomend:
1. Check your DB3.1 cells for competency with the uncut pKDWG2R, and assess the transformation efficiency. If you get less than 10^6 cfu/ug, discard the batch and prepare or purchase a new one. If this is happening, it might be that your ligation is working but the transformation efficiency is so low that you don't get any colonies.
2. Check that the enzymes are working (you have done this, the plasmid is cut in two places).
3. Check that then enzymes are cutting the small insert. Do you have enough (>=6 minimum, https://www.neb.com/tools-and-resources/usage-guidelines/cleavage-close-to-the-end-of-dna-fragments) spacer nucleotides from the enzyme recognition sequence to the 5' end of the primers? This can seriously affect digestion, which in consequence can reduce ligation efficiency.
4. There are many ligation protocols, and it's best to follow manufacturer's instructions. Did you protocol include PEG? When you want to do intermolecular ligation, it is recommended to use a small volume (like yours, 10 ul) and to add PEG, which acts as a 'condensing' agent, facilitating approximation of molecules to one another.
5. Is the T4 ligase OK? Try it with something that you know that works. Remember than ATP is very sensitive to freeze-thaw, so if you have used your ligation buffer very often it might be off (it's best to aliquote it in small volumes just after purchase, and use each twice or three times at most).
All in all, I would make sure that all reagents and reactions are working fine. Once I know that, I would do a 'large' experiment with several plasmid-insert rations (from 1:1 to 1:10 plasmid:insert), do, transformations and grow all the cells on the plates.
Best,
Ruben
https://www.neb.com/tools-and-resources/usage-guidelines/cleavage-close-to-the-end-of-dna-fragments
Hello,
I am not sure how you purified restriction endonuclease treated vector and insert. Previously I had similar problem. Now, I use PCR purification kit for purifying the vector (this avoids UV exposure after agarose gel elecrtophoresis) and use agarose gel elextrophorsis/PCR purification kit. Additionally, I follow 5:1 molar ratio. It works very well.
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