A new plasmid appears on my gel in addition to my construct when I increase the - (Jul/31/2006 )
Hi everyone
I am having a problem with a construct I made. I designed a new construct to be used for a cre animal model. I have tested the construct and verified it. I made glycerol stocks from the verified culture. I am trying to purify my construct in large quantities using a Qiagen Hi speed maxi kit. Whenever I do that I am getting my construct but in addition an additional plasmid appears on the gel (4.5kb). I initially thought that it was a contamination so I replated my glycerol stock picked colonies and made minipreps to verify that they had only my construct. Once that was done I reattempted to make a maxi prep from the new glycerol stock and found out to my surprise that I still got the 4.5 kb plasmid. I am confident that the LB broth is not contaminated I was not able to grow anything out of it even when I culture it for 2 days. I am wondering whether the conditions of the growth (150ml Cx) is leding to such a strange effect. I am also going to reattempt the experiment after I double check my glycerol stock and use a different bench to work on. I just wanted to know whether anyone has had a similar problem or has any helpful suggestions
George Jeha,MD
I am having a problem with a construct I made. I designed a new construct to be used for a cre animal model. I have tested the construct and verified it. I made glycerol stocks from the verified culture. I am trying to purify my construct in large quantities using a Qiagen Hi speed maxi kit. Whenever I do that I am getting my construct but in addition an additional plasmid appears on the gel (4.5kb). I initially thought that it was a contamination so I replated my glycerol stock picked colonies and made minipreps to verify that they had only my construct. Once that was done I reattempted to make a maxi prep from the new glycerol stock and found out to my surprise that I still got the 4.5 kb plasmid. I am confident that the LB broth is not contaminated I was not able to grow anything out of it even when I culture it for 2 days. I am wondering whether the conditions of the growth (150ml Cx) is leding to such a strange effect. I am also going to reattempt the experiment after I double check my glycerol stock and use a different bench to work on. I just wanted to know whether anyone has had a similar problem or has any helpful suggestions
George Jeha,MD
What is the size of the original plasmid? What is the size of the construct plasmid? That is, how does the 4.5 kb plasmid relate to your construct and the original?
What cells have you been growing the construct in? Can you PCR anything from the new plasmid (let's call a spade a spade, and call it a contaminant)? Have you done any sequencing to see what you have?
could it be genomic DNA contamination? I mean, are you sure it is a plasmid??
also, some bacterial strains already contain a plasmid - are you using a normal strain or something a little more exciting?
Are the plasmid samples you're running linearized? If not, it's likely your seeing different forms of the same plasmid (supercoiled, relaxed, nicked, etc.).
I am confident that this is not genomic DNA. it looks like a plasmid witha supercoiled and nicked form and it runs around 4.5 kb while my own construct is close to 18kb. The funny thing is that I do not see it when I purify DNA using a mini prep however it appears with a Hi Speed Maxi prep. I am using DH5alpha cells and will contact invitrogen in order to see whether large preps lead to growth of such plasmid.
George
Hi everyone
I am having a problem with a construct I made. I designed a new construct to be used for a cre animal model. I have tested the construct and verified it. I made glycerol stocks from the verified culture. I am trying to purify my construct in large quantities using a Qiagen Hi speed maxi kit. Whenever I do that I am getting my construct but in addition an additional plasmid appears on the gel (4.5kb). I initially thought that it was a contamination so I replated my glycerol stock picked colonies and made minipreps to verify that they had only my construct. Once that was done I reattempted to make a maxi prep from the new glycerol stock and found out to my surprise that I still got the 4.5 kb plasmid. I am confident that the LB broth is not contaminated I was not able to grow anything out of it even when I culture it for 2 days. I am wondering whether the conditions of the growth (150ml Cx) is leding to such a strange effect. I am also going to reattempt the experiment after I double check my glycerol stock and use a different bench to work on. I just wanted to know whether anyone has had a similar problem or has any helpful suggestions
George Jeha,MD
What is the size of the original plasmid? What is the size of the construct plasmid? That is, how does the 4.5 kb plasmid relate to your construct and the original?
What cells have you been growing the construct in? Can you PCR anything from the new plasmid (let's call a spade a spade, and call it a contaminant)? Have you done any sequencing to see what you have?