RF-Cloning.org Forum

Anything and everything cloning: Go...

You are not logged in.

#1 Re: rf-cloning troubles » No colonies & no band on agarose gel after 2°PCR » 2015-10-27 15:58:34

Hi Steve,
firstly thank you for your feedback and suggestions. I really appreciate the time you take to maintain the site and answer questions on the forum.
I just wanted to give an update- I tried using DMSO in my reactions and I also used the GC buffer (this is an extra buffer from the Phusion HF DNA Polymerase Kit made for PCR of GC rich sequences) and got a bunch of colonies this time.
Unfortunately none of the 25 clones I screened contain the promoter I was trying to clone in but rather it appears that they all still have the parental promoter.
I digested my 2°PCR product for 2 hours with 20U DpnI digestion at 37°C, followed by 20minutes at 80°C, so I dont know why I still have parental plasmid. Is there something else I could do to make sure all my parental plasmid is digested?


#2 rf-cloning troubles » No colonies & no band on agarose gel after 2°PCR » 2015-10-19 09:59:47

Replies: 3

I am trying to replace the Probasin promoter in the p255 parental plasmid with another promoter, however no complete sequence is available for this parental plasmid and so far I have only sequenced the portion of the plasmid where the Probasin promoter is located. The p255 plasmid is approximately 8-9k bp and my insert is 550bp. The 1° PCR appears to work well and I can gel purify sufficient amounts of my ~600bp megaprimer, however after 3 attempts I am still unable to see either a correct band after running my 2° PCR product on an agarose gel or any colonies after transformation.
For my first attempts I used the PfuUltra HF DNA polymerase (@2.5U/ul) and the following 2° PCR protocol:
Denature @95°C for 2mins; 15x [Denature @95°C for 30s; Extension @72°C for 10mins]; Final extension @72°C for 5mins.

I then switched to the Phusion HF DNA Polymerase (@0.4U/20uL) for the my last attempt and I used the following 2°PCR protocol for a total of 100ng parental plasmid & 150ng megaprimer (in order to have a molar insert:vector ratio of 20):
Denature @98°C for 30s; 15x [Denature @98°C for 8s; Extension @72°C for 4mins]; Final extension @72°C for 5mins.

I have tried transforming both self-made chemically competent Stbl2 basteria and commerically bought OneShot Top10 bacteria on ampicilin-selective plates with 1ul of the DpnI-treated 2°PCR product per 20ul competent bacteria.
The only good thing so far is that I do not get colonies for my 2° PCR negative control.

Any idea what is going wrong here? Should the 2°PCR be modified?

Board footer

Powered by FluxBB