I tried cloning IRES-EGFP region to my vector. For the first try, I try with PCR cloning (Q5) for getting the IRES-EGFP region. I tried to do ligation and transformation (DH5 alfa), it worked well and I got my desired band.
The problem arise after sequencing, there are missing base in the IRES region.
So, I tried second times, using direct cloning to change the (mutation part). Unfortunately, the sequencing didn't work and my Prof. told me to tranfect the plasmid into the cell ( I used 293FT cell). The result showed there's no GFP signal, means that my plasmid somehow wrong.
I tried the third strategy, using one enzyme cut for the vector and two enzyme (double digest), and for the vector I used FastAP. But in the end I got less colony than the two strategy. What do you think goes wrong with my cloning?
I really appreciate your help
Have a good day
P.S. : I got a weird result from the same plasmid and the same enzyme. I cut the plasmid first with NruI and PvuII (separately) for 1 H. It showed more than one band, the band supposed to be only one. The next day I tried the same, with less plasmid and less enzyme (ratio still the same), and longer incubation time. But I got the result I want, only one band appeared on the gel.
What do you think happend?
Dear Nurul,
For your first question, did you sequence the IRES-GFP plasmid used as template to confirm that it does not have missing bases? Are these missing bases located within the amplicon or in the extremes where o close to the primer binding regions? If these missing bases impair the binding of the ribosomes to the IRES then it is normal not to see GFP expression.
For the second question, I gues you had an incomplete digestion in your first attempt and what you saw were the different conformations of your plasmid (cut, nicked and supercoiled) plus the excised fragment. In your second attempt the digestion was completed and then you only saw the cut vector and the excised fragment.
Hope it helps,
Dear Mr. Dupraz,
Thanks for your feedback, I already found what's when wrong with my PCR clone. The problem is not the PCR product but my vector NTI file, something wrong with my sequence data. So, when I compared them, they just didn't match.
The missing base pair located like 20 bp from primer binding region.
And for my third attempt, I got the desired plasmid and got the GFP signal in the cell.
Best,
Nurul Fadhilah
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