I am doing this for the first time and I need a quick check wether i have understood the method correctly.
My plasmid has a CAM gene which is going to be deleted and replaced with KAN.
1. Primer A -vector , is a reverse complementary and starts after ATG og CAM (the gene which is going to be deleted)
2. Primer B -vector is complementary (forward) and starts after the Stop codon of the region which will be deleted (CAM)
3. Primers for my gene of interest F and R starts from ATG to STOP
4. The Forward primer of GOI (C) has 16 n complementary to primer A (of the vector) on its 5-end
5. The reverse primer og GIO (D) has 16 n complementary to primer B (of the vector) on its 5-end
A+C and B+D ?
Best,
Hi
I am little bit confused with your description.
Please check the following attached article which is under press (Manoj Baliram Pohare and Mitsuru Akita, 2016). in this article also, primers are designed in the same way as that of fast cloning with simple explanation in the form of figure. Please check Fig. 2. Schematic diagram of in vivo HR-based cloning.
This figure will clear all your doubts about the primer designing of such methods.
Thank you, I couldn't find the article , can you please provide the title.
Thanks
Thanks, its not available online yet, in press.
Give me your mail Id, i can forward you my article.
Hi,
You primer design seems ok to me. You have to amplify your vector with primers A and B and your insert with primers C and D.
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