My vector size 5.1kb and insert size is 4kb. I performed double digestion of vector and insert, ran them on the gel and then extracted them from the gel. 260/280 ratio was good. Then I did ligation using T4 ligation at 4C and transformed into E. coli DH5a. After 24hr, I did not see any colony. I left the plates for another 2 more days in the incubator. Now I see some tiny colonies. My question is -
1) Is there any way to check whether ligation or transformation is the problem?
2) The OD600 of competent cells was about 0.9. Would that be an issue?
3)Are the tiny colonies after 3 days transformed colonies or not?
4) Any suggestion for successful cloning with my vector and insert sizes?
Thanks in advance
there can be several reasons for not having colonies, perhaps the ligation did not work or the transformation was not carried out correctly or your host cells are not competent.
Concerning the colonies which appeared a few days later, they are false positives, they appeared either because the antibiotic lost its functionality in your culture medium, or it was spontaneous mutants which appeared.
The easiest thing to test is your competent cells, just use a small amount of pure uncut plasmid DNA (it can be your vector), maybe 10ng or so, and do a transformation to see how many colonies you get. You should have thousands or tens of thousands.
A second possible problem is that you did not recover your plasmid or insert after gel purification. Did you quantify your recovery?
A third possibility is that the DNA was damaged, if the gel was exposed for too long to the UV light you can crosslink the DNA and it won't be viable.
Lastly it might be the ligation didn't work, but this is rare if your ligase and buffer are reasonably fresh.
Dear Tarique vai, Please make super-competent cells using modified hanahan method, it will work better.
K. M. Kaderi Kibria Thanks for your suggestion. Can you please share the protocol that you are using?
Michael J. Benedik I am not sure, what you meant by recover plasmid/insert after gel purification. I quantified the amount of vector and insert. Could you please elaborate?
Abdullah Al Tarique to determine what went wrong, you need to include proper controls. For example, uncut vector will be control for transformation efficiency. Single cut and ligated vector will be control for ligation efficiency. Cut vector without ligase will be control for background colonies from inefficient digest and/or self-ligation (this is not of your concern now, but it's good to keep all controls everytime).
https://bitesizebio.com/10273/pin-pointing-dna-ligation-problems/
BTW is the OD600 of 0.9 during competent cells preparation? That is way too high. Depending on temperature at which you grew your cells, it may be of high problem.
(cells grown at 37°C are very sensitive to proper OD600)
Tomáš Hluska While preparing competent cells, if the OD becomes 0.9, should I dilute them or prepare them again at OD 0.4-0.5?
Abdullah Al Tarique no, it's not about concentration of the cells (with that would the dilution help), but about the state of the cells. If they reach high OD600, it means they are late in their growth phase and their competency decreases.
BTW what is your method of transformation? Electroporation or heat shock?
Abdullah Al Tarique for heat shock, the cells are sensitive to OD600, especially if you grow them at 37°C (see Fig. 3 in this paper Article Inoue, H. , Nojima, H. & Okayama, H. High efficiency transfo...
). So next time, grow them at lower temperature and check the OD600 carefully.- Badges
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