Problem in picking single colony
Posted 22 July 2011 - 05:56 AM
I am currently cloning an leukimia gene which has its wild type allele and two mutated types which are both of larger base pair in size.
And i have recieved a blood sample which have all the above three alleles.Then i use its DNA to do a PCR and have confirmed the above three alleles.
Then I use the PCR product to do a ligation process with pGEMt vector.After that, i transform the vector into DH5alpha positive competent cells Then i use the DH5alpha positive competent cells to spread plate.
Afterwards, i pick single colony from the plate and i am confident that i have picked one colony each time only since there were toofew colonies for me to make a mistake.
After that, I put the colony in LB medium and shake overnight at 37 degree.
After one day, i directly use the LB medium with those competent cells to do a PCR since i belive that 94 degree in the denaturation process can directly break the cell membrane of those cells and release the DNA. Thus i can skip the miniprep process.
Unluckily, i have found all the three bands shown up in a single PCR product. And i think that is impossible since a single competent cells could only pick up one vector and that vector should have only contained one allele each. Thus a single colony, after a pcr, should have shown only one of the three allels as mentioned above. And it isvery frustrating for me to get all the three alleles together in one single colony.
Pls anyone help me!thX!!
Posted 22 July 2011 - 06:37 AM
Posted 22 July 2011 - 05:04 PM
As i am still an undergrad, i dun know whether my above concept is rite. anyway, thx for your detailed reply