marker swap plasmid
Posted 28 November 2009 - 03:25 PM
There are so many experts here. I like this forum.
Recently, I have to switch the HIS3 marker (which is used to disrupt the gene I am working) to the KanMX marker. I was wondering how i can verify the correct colonies after I transform the plasmid with KanMX flanking the HIS3 gene sequences. Do I have to do colony PCR to test whether the kanMX marker is inserted in the gene of my interest like the standard gene disruption protocol? I am thinking that if the transformant doesn't grow on SC plates (no histidine), then the HIS3 gene inserted in the gene of my interest has been disrupted. Could anybody tell me if I am right.
I am looking forward to getting your kind reply and thanks for your time to read and help in advance
Posted 30 November 2009 - 11:59 AM
In my lab, I am the only person who needs to work on yeast genetics and no body else in my lab has done something related to yeast genetics. Nobody in my lab can help me. I have to read a lot and download protocols online, then follow the protocol to do experiments.
I read some papers about gene disruption in yeast. In these papers, people verify the correct deletion colonies by colony PCR. Unfortunately, when I did colony PCR to verify, no products were obtained. I realized that I don't need to do PCR since the colonies do not grow on SC-plates (no Histidine). It means that the HIS3 gene has been disrupted. Could anybody kindly tell me if I am right?
I really appreciate your help in advance.
Posted 30 November 2009 - 03:41 PM
Posted 01 December 2009 - 03:06 AM
You could also sequence the plasmid to see if there is a disruption.
Thank you so much for your good suggestion. I got the plasmid from the other lab. I didn't check the sequence. I will do that tomorrow.
I have already transformed the plasmid and I got several colonies which can not grow on SC-his plates. This is the first time to do marker swap. I don't know how i can verify the correct colonies. I know that there is chance that the KanMX gene also insert into the endogenous his3 gene since the strain I am using has endogenous his3 gene with only a point mutation. Do I need to also verify that the KanMX is not inserted into the endogenous his3 gene.
Thanks a million for any comments and advice.