RF-Cloning.org Forum

koikkarah93

Member

Registered: 2019-07-04

Posts: 1

Issues with the Secondary PCR reaction

I have been attempting to do RF cloning method to insert a 70 bp sequence into a 6kbp vector.

The Primary PCR worked well with the required segments obtained.
The issue has been with the secondary PCR.

The procedure I used is as follows:
a. 100 ng of the vector
b. 1:20 ratio for the Insert.

I used the 2 step PCR method with an annealing temperature of 72C for 30 seconds since the portion I wanted to amplify in the Secondary PCR is nearly 1kB (70 bp from the insert and the rest from the Vector). The incubation with dpnI was done at 37C for 2 hours and then heated at 80C for 20 mins to deactivate it.

I used Mac1 one competent cells to transform the DNA. I used 2 ul of the mixture in 100 ul of competent cells.

However I could not observe any growth of colonies on the bacterial plates.

I am attempting this method for the very first time and would truly appreciate any assistance regarding my situation and if there is a mistake I have committed, please do point that out as well.

Amit

Steve Bond

Administrator

Registered: 2014-01-23

Posts: 130

Re: Issues with the Secondary PCR reaction

Hi Amit,
I think you may have miscalculated how much of the vector needs to be replicated in the reaction, unless you are trying to delete 5 kb? If you can give me a little more information about your sequence, I can retrieve the project from the database and have a look.
Otherwise, your protocol looks pretty good to me, and the small insert should make things easier. I have high hopes we'll be able to sort you out
-Steve