RF-Cloning.org Forum

Hello

I am using this technique to construct a plasmid but I am facing several dificulties.

I am using a primer design already published, and so, not designed in the website. However, later, I used the site to design the project and the primers the program gave are equal to the ones published.
The MPr are approx. 7800bp and the plasmid 3600bp. The insertion deletes a region of about 1200bp and the 2nd PCR amplifies about 2400bp. The final plasmid will be 10200bp.

The first PCR, for Megaprimers, is working properly. Then, I used several conditions for the 2nd PCR (2 step or 3 step 52/55/60ºC; 100 ng to 1800 ng of MPr and 50 to 100 ng of plasmid), including controls. The controls I used were with or without each component of the PCR mix. After that I ran the products in gel to check if there is amplification of the new plasmid. The conditions I used were similar to the ones described in the website and in the papers. I use Phusion High-fidelity polymerase from Thermo.

The problems are:
1) The MPr is supposed to disappear in the 2nd PCR but instead it seems to be amplified. I have a band in agarose stronger than the band corresponding to the initial amount added in the PCR mix.
2) All controls with or without phusion, etc... seem to work as the correspondent band of the resultant plasmid appear only in the complete reaction (although the MPr does not disappear). I never checked but I believe the band appearing in the agarose seems to be the correct size of the resulting plasmid that I want.
3) DpnI digestion after the 2nd PCR works on every control (with or without each component of the PCR) as colonies (about 200) appear only after transformation with the complete reaction, and no DpnI gives confluent growth.
4) The initial plasmid has two selection markers. One of them, Amp, is removed after the insertion. So first, I screened the colonies by replica plating searching for colonies susceptible to Amp. The majority are resistant to Amp but in 50 colonies tested, normally 10 are Amp_S. Then I tested these colonies by PCR for the presence of the insert and they are always negative for the presence of insert (appropriate controls included).
5) Another hint that in most of the cases the 2nd PCR is working is that I can perform a third PCR (inverse PCR) to delete part of the region that was inserted in the 2nd PCR.

Any ideas?

I think I did not save the project but I copied it to a word file. I can post it here.

Thanks in advance for the help
Filipe Almeida