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I perform my PCR on 25 ul incubation and I ALWAYS heat a mixture containing cDNA, primers and PCR buffer (12 ul volume and 1,5 ul buffer) for 1 min at 95°C and cool it on ice for 2 min. I then add 13 ul containing MgSO4, buffer, dNTPs , Taq polymerase (usually a mixture of 0.1ul Taq recombinant + 0.02 Taq Hot start ThermoFisher). If the PCR is particularly difficult I rise the Ta up to 60, 63°C.......It usually work

I am also having this problem trying to amplify CAG promoter with intron. My sequence is 68% GC rich and just over 1600bp long. We have tried Taq and Q5 polymerases, designed several new sets of primers, tried different starting concentrations of plasmid DNA, different Mg etc. I am running low on options too. I am interested to see if anyone else has any solutions to this problem!

Same thing here. I was trying to PCR amplify the 1.6 kb pCAG that is 67% GC, which contains the 1010 bp "chimeric intron" that is 74% GC, using Q5. Adding the GC enhancer in the Q5 kit did not solve the problem. My current solution is to go back to restriction enzyme-style cloning whenever using pCAG.

Here is another choice in case if you love Gibson Assembly as I do. The pCAG-containing vector can be linearized by restriction enzyme digestion, and directly used as one of the fragments in the assembly reaction.

Hi there, I am having this exact same problem trying to clone from the CAG promoter. Did you ever resolve this issue?

Hi all,

I also struggled quite a lot trying to amplify this promoter. At the end I managed to get enough amplification to perform its cloning. Normally, I used the full plasmid containing the CAG as template in the PCR and it never worked. Using just the digested CAG promoter as template gave me better results.

I used Q5 master mix (addition of GC enhancer did not improved the result).

I used primers with 15 bp homolgy and 20 bp overhangs(that's why the first shor cycle at 48C).

My settings

1-98C > 2'

2-98C > 30''

3-48C >15''

4-72C >50'' goto 2 x 5 cycles

5-98C > 30''

6-70C >15''

7-72C >50'' goto 5 x 40 cycles

8-72C > 5'

9-4C > 2'

Hope it is of some help.

Cheers,

Hi Sebastián Dupraz,

I have the same problems of amplify the pCAG promoter for my Gibson assembly. Exactly followed Sebastián's PCR profile, but no luck for me at all. Has anybody tried Sebastián's PCR profile as well and got the good results? Or any other suggestions for PCR of pCAG? I am going to try touchdown PCR and other enzymes like KOD.

Dear Zhenghui,

As I said before I repeated 3 times the PCR with those settings, pull them together and I got a faint band. It was luckily enough to achieve its cloning.

However, one of my colleagues mentioned to me another, I think clever, strategy. This was to amplify the vector backbone you'll use with primers having overhangs matching the sequence of an restriction-enzyme-digested CAG promoter from another source. Even is your plasmid backbone is over 6Kb it should be easy to amplify it without errors using phusion or Q5 kits. If you need me to expand this idea to make it clearer let me know.

At the end it may be faster this way than endless trying-failing cycles.

Cheers