Hi I am just an ordinary university student that is currently studying in a lab to graduate. I have not been long since I have been studying in the lab. Now I am learning how to make a vector and insert DNA and perform ligation and transformation. I have been doing this for 4 times.
However the point of the problem is when after i conduct ligation and transformation and spread it into the LB plate, alot of colonies come out of the plate.
The plasmid DNA i am using is pBSK- (vector)and T-easy(insert). In four LB plate I conduct the spreading by vector only/inset only/1:2(ratio of vector and insert, vector1inset2) /1:4
If it is performed right there should be no or little colonies in vector only and no colonies in insert only and right amount and size of colonies in the rest of the plate
However in all my plates there are alot of colonies. Also alot of satillites.(It looks as if it is contaminated. All of my lab students(they came into the lab with me) have the same problem
I searched alot of troubleshooting cases and changed alot of factors that could affect the result(water, new plate, new medium, check mistakes in protocol) also conducted in the way our professor said to us. But all the results of three university students have the same results.
Our professor and other research assistents and lab people is getting confuse of our results because it hasn't happen to them before...... we are all getting stressed about it. What might be the possible cause of problem?????
If you have multiple colonies in the inserts only plate, it means that you may have problem with your antibiotic:
- did you put any antibiotic in the LB plates for spreading?
- are you using the correct antibiotic? you should use the antibiotic that your vector is resistant to
- Is the concentration of antibiotic correct?
- LB plates with antibiotic used for spreading should be prepared fresh or the day before spreading to ensure that the antibiotic in the media is still working
Heesu Jeong What antibiotic are you using to select cells?
I think your vector has Ampicillin resistance. Ampicillin stock solution is not stable for long. I prefer making fresh Ampicillin. Also, LB+Ampicllin plates stored for long is not good, so try using fresh plates. You can check the plates for ampicillin resistance by simply streaking the E.coli cells used for transformation. If the cells grow, then the problem is with your LB plates.
I would also suggest you to make new plates with new Ampicillin stock. Whenever everyone in the lab has a such a problem, its mostly the LB plates.
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