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cloning size is not correct


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#1 kaykwli

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Posted 18 August 2004 - 06:17 AM

I have used the Invitrogen TOPO cloning kit to insert my DNA. After picking the white colonies and did the PCR trying to confirm DNA has been inserted in plasmid. Although I did find the expected size in band on agraose gel, I also see a smaller band in size between the plasmid size and the plasmid size plus my DNA. What is the problem?
If I tried to sequence my inserted DNA, will I see a mixture of DNA sequence?

#2 amira

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Posted 18 August 2004 - 06:36 AM

I have used the Invitrogen TOPO cloning kit to insert my DNA.  After picking the white colonies and did the PCR trying to confirm DNA has been inserted in plasmid.  Although I did find the expected size in band on agraose gel, I also see a smaller band in size between the plasmid size and the plasmid size plus my DNA.  What is the problem?
If I tried to sequence my inserted DNA, will I see a mixture of DNA sequence?

you mean that after your PCR on 1 clone you have 2 bands?
did you do the PCR with the primers specific to the plasmid (and not tha same than your PCR)? In this case it's mean that you picked more that one clone.

#3 kaykwli

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Posted 18 August 2004 - 06:41 AM

I have used the Invitrogen TOPO cloning kit to insert my DNA. After picking the white colonies and did the PCR trying to confirm DNA has been inserted in plasmid. Although I did find the expected size in band on agraose gel, I also see a smaller band in size between the plasmid size and the plasmid size plus my DNA in the same clone. What is the problem?
If I tried to sequence my inserted DNA, will I see a mixture of DNA sequence?

Edited by kaykwli, 18 August 2004 - 06:42 AM.





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