2 replies to this topic

[Rico]

hi everyone, i am new here.

i did the blue white screening for my ligasion, only several colonies(less than 15 colonies), white, grow on the plate. i picked the white colonies and grew them in fresh plates(LB+Amp). Then did the colony PCR to check if my target fragment was inserted. According to the colonies, white, resistant to Amp, i thought my DNA fragment shoule be inserted in, but, no, nothing:(((

i will be very appreciate if someone could explain what's the white colonies, and why they can grow on the LB+Amp and behave white color without my DNA fragment inside (the ligasion is double enzyme digested pcr fragment and also double enzyme disgested plasmid, NOT pGEM-T...).

thanks a lot!

[HomeBrew]

Did you have a positive PCR control?  How can you tell the difference between no PCR signal due to no insert versus being due to a failed colony PCR (colony PCR fails quite often)?  Check a few by plasmid isolation and RE digestion...?

[Rico]

HomeBrew, on 22 February 2011 - 07:25 AM, said:

Did you have a positive PCR control?  How can you tell the difference between no PCR signal due to no insert versus being due to a failed colony PCR (colony PCR fails quite often)?  Check a few by plasmid isolation and RE digestion...?

Thank you, HomeBrew. I did the plasmid isolation finally, as well as digestion. However, all the colonies are not correct ones. I guess the ones are some small fragment generated by PCR(although i did the gel purification, but maybe still some non-significant fragment left), inserted into the target plasmid...

whatever, i did another round. Thanks a lot!!