1 reply to this topic

[EbolaZaire]

Hi,

I'm doing TOPO TA cloning and think I set up my positive control using the pUC19 plasmid wrong.  In the vector ligation reaction I simply replaced PCR product with the pUC19 plasmid; is this correct?  Nothing grew on my positive control plate, but colonies grew for my samples and nothing grew for my negative control which leads me to believe I didn't set up the positive control correctly.  Help?

[bac]

Hello,
if I got your message right, you used pUC19 as insert with a TOPO vector (which one? just out of curiosity).
Then well yes, I think you set the positive control wrong. It would be hard to get ligated a circular plasmid (pUC19) into the TOPO vector, so I would not be surprised that you had no colonies. What about the actual cloning samples?

pUC19 is provided in the kit for two reasons: first to check the transformation efficiency of your competent EColi*. In this case you would have only to transform pUC19 into competent EColi (not to set up the cloning reaction). Then I think you can use it as positive control for M13 PCR  if you want then to screen the colonies having the right insert.


* see p 18 in this manual http://tools.invitro.../topota_man.pdf
Note: I provided the link just as reference, it can be that this manual is not of the same kit you are using, there are several TOPO TA kits!

hope this helps