I am working on molecular biology and vaccine technology. I am repeatedly facing a issue in Ligation transformation for which I have tried troubleshooting it by many ways. By increasing or decreasing the heat shock time i.e., 45seconds to 90seconds, also concentration of Ligation mix optimization, 12.5ng-10ng-5ng three of these concentrations checked. but still it is not working. I am not understanding why is it happening. I was also thinking if the efficiency of my competent is less, i.e., 106 then for 1ng this gives at least 29 colonies. then I am using more than these concentration. I really do not understand what else I can do. I have used 1:3 Ligation molar ratio for my Vector : insert. Should I be using a different ratio and check or is there any other way that I know where I am going wrong. My vector and insert double digestions are proper and clean. I have rechecked it again and again. I really think there is a problem in transformation only. Can Anyone help me please.
Hi,
1. Probably you can use an undigested vector as a control to see if the problem is with transformation or ligation?
2. If you get correct colonies by transforming the undigested vector, then maybe the issue is with ligation. How old is your ligation buffer, if it was thawed/frozen several times, the ligation efficiency might be reduced?
All the best!
Hi,
It is of course necessary to mention that every system might be different considering the vector size, competent cells and the quality of your mix buffer as Pruthvi Balachandra Kalyandurg mentioned above. Perhaps the time of incubation after the ligation reaction is not enough. In my experience I tried with up to 6 h at 16ºC (according with the manual specifications) to ligate a 3kb insert with no positive results. It only worked after I added an extra step of 5-6 days in the fridge (4ºC) before transformation of chemocompetent cells. Even when I obtained only a few colonies, all of them contained the ligated product. Commonly it shouldn't take this long, but in principle slowing down the interactions between vector and insert seem to show better results. As I mentioned every system is different, but this is something you could try.
Regards.
There are two major limiting steps - the ligation step is critical. Increasing more insert always helps - indeed, we routinely do higher vector/insert ratios and see best results with higher insert concentration. While lowering ligation temperature (especially for blunt ends) and increasing duration helps, there are so many new ligase products now commercially available that can do the best job within an hour.
The second often ignored step is competency of E. coli cells. Most people use plasmids to test efficiency of competent cells and are satisfied when they get few colonies. But this doesn't truly measure transformation efficiency. The best test is to ligate is to use a cut plasmid and test self ligation. Efficiency of transformation is much lower with re-ligated plasmid than closed circular plasmids.
Hey, Thank you all, I have tried ligation again with positive controls like single digested plasmid ligation which has to happen if ligase is working well. And my ligation this time has worked well when I have increased the concentration of ligation mix in the transformation. Ideally in troubleshooting it was given to reduce the ligation mix as it inhibits transformation, but for me increasing its concentration really worked. Thank you all for your suggestions.
Hi Kotwal,
1. What was the temperature, you had used in heat shock time? i.e 40 degree C
2. Which type of cell had used in the experiment? i.e. member of eneterococcaceae
3. What will be the size of the vector and insert? i.e insert 1 kb vector 3 kb, then you will be used 3:1
4.Which chemicals you had used in the preparation of competent cells? i.e, CaCl2
5. What was the incubation time for the preparation of competence?
I want to know the answers to help you in my way. In this experiment, you should have used more than 10 ng ligated mixture.
Hai Rajarshi,
1. Heat shock was 42 degree 45sec once next I have used 90sec the other time.
2. You mean for transformation. If so it was E.coli-DH5 alpha.
3. My insert is 1kb and vector is 6 kb
4. CCMB buffer was used.
5. I did not understand the last question.
I have tried with different concentrations of ligation mixture. 5ng, 10ng and 12ng as well. But 3 of these concentrations did not work for me.
Hello Kotwal,
1. Heat shock will be at 42 degree C for 40 sec and immediately put the tube into the crushed ice for 5 min then put it at 37 degrees C incubation for 2 hrs.
2. Fine, you will use CaCl2 for the preparation competence
3 Insert:vector:: 6:1
4. What stands for MB, CC means calcium chloride. I think, don't know, please elaborate CCMB. Needs calcium chloride buffer only, But the growing medium is not simple LB, a little bit with different composition.
5. During the preparation of competence, You should have to incubate your cell in calcium chloride. That's why I ask you to know how long you incubate.
Thank you, feel free to ask me.
CCMB stands for the composition of Cacl2. 2H20, MnCl2.4H20, MgCl2. 6H20, Glycerol 10%.
And I incubate the cells with CCMB buffer for 20min.
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