I am a graduate student doing sub cloning of two similar genes into pBC SK(+); one of the gene was inserted but few colonies appeared on the agar after transformation, the other is not inserting into the plasmid no matter how I elevated the ration of insert to plasmid.
What is the explanation?
Hmm, lots of possibilities. Cloning works well on paper but lots of things can go wrong. Here are some options.
1. The ligase is not working. Either the buffer has expired (this can happen, especially if it has been frozen and thawed repeatedly) or the enzyme is inactive. Get a new ligase kit to try again!
2. Insufficient DNA amounts. The usual ratio of 3 moles insert to 1 mole of vector is based on 50 ng of vector for every 3 KB of vector size. So, if your vector is larger you'll need to scale up the DNA (if your vector is 9 KB you need 150 ng per ligation reaction). Double check the math!
3. The cells aren't competent enough. Generally, cells are good at -80 for 1 year, then they start to lose quality. Try transforming a positive control along with your ligations.
other options . . .
try a different sort of ligase and ligation conditions (some work well at room temp, others at 16 degrees)
ask a colleague to try it out for you (sometimes that can help), the joke in some labs is that you give it to the newest person
troubleshoot all your digestion enzymes to make sure all are actually cutting your DNA
ensure no ethanol carryover from DNA purification as that messes up ligations
hope this helps and good luck! Cloning can be really frustrating
Hello, Dr, Klocko
Thank you for your response. I tried the enzyme in a different experiment and it's working fine and I also ran the digestion on gel. I also used new electrocompetent cells, DNA amounts and incubation periods were modified repeatedly.
The ethanol makes sense because the gel extract was not of that pure quality.
Best regards!
Hello Heba,
Sounds like the troubleshooting is narrowing down possible issues. Depending on the gel extraction kit, you can also get bead/resin carryover (column kits don't have that issue but slurry/bead kits can). Just add in an extra spin and tube transfer to the elution step (add elution buffer, incubate, spin, move to new tube, then repeat the spin and tube move).
Even if you get just a single colony it could still be what you want! I've had that happen twice.
Hello, ethanol can have an effect, as well as other elements that have already been mentioned above. If you are using some carriers in your fusion such as glycogen it can also interfere with the binding.
If you have already tried everything else (as you told us) perhaps you can try cloning directly into plasmids prepared for direct cloning such as Topo.
- Badges
- Science topic
- Similar topics
- Agricultural Science
- Agronomy
- Crop Science
- Crop