4 replies to this topic

[Teagan27]

Hi,

I am attempting to linearize a plasmid by PCR.. however.. i am getting no amplification what so ever. There is about 50bp between the primer annealing sites. I have checked the primer sequences and orientation, as well as my PCR protocol (my supervisor has double checked all this). The only other option i have thought of is  perhaps linearizing the plasmid (i.e. in the middle of the MCS by restriction digest) in the middle of where the primers are supposed to bind before doing the PCR, as steric hindrance may be a problem. Any help or hints would be appreciated.

Cheers Teagan  

[jiajia1987]

Why are you linearizing your plasmid?  For deleting a portion in the plasmid and obtaining a linear piece of DNA? Or for other purposes?

[perneseblue]

Teagan27, on Aug 12 2009, 02:50 AM, said:

The only other option i have thought of is  perhaps linearizing the plasmid (i.e. in the middle of the MCS by restriction digest) in the middle of where the primers are supposed to bind before doing the PCR, as steric hindrance may be a problem. Any help or hints would be appreciated.

Cheers Teagan  

Yes, linearising the plasmid before PCR would help.

It would also be helpful if you should tell us the conditions and reagents that you are using to conduct the PCR reaction. You might need to optimise the PCR conditions. How big is your plasmid and what polymerase are you using? What is the structure and sequence of your primers?

I am assuming that you are using this method to introduce novel restriction site into the plasmid. If so, just as a reminder, the tm of the primer is calculated based only the DNA segment that actually binds to the plasmid template. The rest of the primer is not counted. eg A primer is 100bp long, however only 24bp binds to the template. Thus the tm of the primer is calculated based on that 24bp sequence.

[Teagan27]

Hi,

Thanks for replying.. i am using PCR to linearize my vector as i am using the Infusion PCR cloning kit to constuct two vectors. The kit relies on very little back-ground of vector which has not been double digested as there is nothing to stop the vectors from re-ligating (i.e dephos when using ligase).

I tried to do double digests with restriction enzymes (invitrogen) (my protocol was reviewed several times  but the back-ground was high). So the company suggested that i linearize the vector by PCR to reduce the back-ground. I was using Invitrogens Platinum High fidelity Taq. At first i did not add the magnesium to the PCR mix (as a fellow student in the lab said he did not use it and it worked fine). I thought i would try it anyway. And guess what? i got nice amplification last night

Problem solved.

Cheers guys.

[phage434]

If you have grown up your plasmid in a dam+ strain, you can eliminate the plasmid by cutting with DpnI, which will cut the GATC sites from the plasmid, while leaving the PCR fragments (which do not have a methylated A) uncut.