Currently I do pENTR D TOPO cloning. I transform my construct (plasmid containing my interest gene) into E. coli DH5 alfa and then checking the result by doing colony PCR using KAPA. I use M13 forward primer and my candidate gene specific reverse primer.
The colony PCR result is quite confusing. Several colony only get 1 band (the size is correspond to my candidate gene), this is the good result. However, the other colonies show more than 1 band (It contain 1 band that correspond to my gene and also the other bands).
So far I only continue my work with the colony that show 1 band only. Do you think it is worth it if i check also the other colony that contain more than 1 band? Why this is happen? Any comment will be helpful. Thanks
if you post the band sizes and the expected band size it may become clearer but one thought is that you have too much insert which has ligated then inserted 2 copies into the vector. I would continue with checking the single band product which is probably the correct thing and leave the double band as an interesting but irrelevant problem but sequencing both bands would probably make the answer clear
There are several possibilities;
-If your primer sequence appear in vector sequence you may get multiple bands. But I am sure that is not the reason.
- check your primers for misspairing and 3' stability. if your second bands are 20-40 bp higher your original bands your primers might be annealing with your pcr product and creating second band. or your pcr templates might be connecting each other.
- kapa kit contains a high performance taq polymerase. it can cause nonspecific bands with vector pcrs on low temperatures.
try this;
- increase your annealing temperature around 5 degrees celcius (thinking your amplification temp is around 55)
- decrease extension duration to half.
- and for last step, you can try to add dmso to your reaction solution like around 5%.
thank you for your advice, that is really helpful @PaulRutland @DenizKom
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