I am replacing GFP with TDtomato for my sgRNA KI construct. First, I performed a PCR for TDtomato using a TDtomato plasmid as the template. When I ran the gel, I observed two bands (700 bp and 1431 bp). This is because my reverse primer has two binding sites. I then purified the 1431 bp band using a gel extraction kit and confirmed its size by running another gel.
Next, I assembled the purified 1431 bp TDtomato fragment into the pLL3.7 vector. I had already digested the vector with EcoR1 and NheI to remove GFP and create sites for inserting TDtomato. After running the gel and purifying the digested vector, I used both a seamless cloning mix and traditional ligation methods to assemble the TDtomato fragment into the vector.
After the transformation, I obtained clones and performed colony PCR. However, all clones showed a 700 bp insertion instead of the expected 1431 bp. Why is this happening?Your suggestions will be highly appreciated. Thank you
Dear Muhammad Shoaib ,
I assume that you are using the same primer set for your colony PCR as for the initial cloning, so the 700 bp fragment might be expected and might have an advantage (check for example elongation time) in the PCR over the 1431 bp amplicon.
You should pick one or two of your 700 bp clones, mini prep the plasmid and digest it with EcoRI and NheI.
Best wishes Soenke
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