Hi all,
I have a linear fragment having overlapping ends. I wanted to know that whether by Gibson, can we circularize that linear fragment and use it as a rDNA for transfection or not?
If possible, kindly mention how?
As Gibson reaction requires the master mix, enzymes, vector as well as insert so how to modulate the protocol and whether it will give the expected result or not?
Hi,
I guess as long as both ends have complementary regions for each other, the fragment has the origin of replication and a bacterial selection marker, Gibson method should work.
What is the total length of fragment? Just make sure both ends have 15-20 nucleotide overlap/complementary region. You can try with 100ng of DNA in one single reaction. Just add the DNA, 2X Gibson master mix (So it could become 1X), and makeup volume with deionized MQ water. Do not use TE buffer or any salt in this reaction as they influence the enzyme efficiency. Keep it at 50 degrees C for 15-30 mins and transform all of it into competent bacteria.
Later you can screen your colonies, directly by PCR and Sanger sequencing.
All the best
@Mohd Ahmad thank you for your reply. I have 40 bp of overlapping ends and fragment length is 10kb.
I'm planning to use 1X master mix and rest of it as my fragment making total reaction volume of 5ul.
That's nice. I would recommend to try atleast 10ul reaction. Let me know, how did it go for you.
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