I have made a construct including 2 PCR produced inserts and a CMV-EGFP-bGH poly A between them.
At first, insert 1 was created by PCR and cloned into pSL1180. Cloning was confirmed by conlony-PCR and restriction analysis was done to check the construct.
The same process was repeated for insert 2.
After that, EGFP sequence subcloned from PEGFP-N1 into pCDNA3.1. Then, CMV-EGFP-bGH poly A subcloned into the final construct. Insertion of sequences was confirmed by colony-PCR.
Constructs including 1and 2 inserts were confirmed by Restriction analysis.
I transfected this final construct into HEK293 to check EGFP expression and it was OK.
What do you think about an extra band with the same size in restriction analysis with a single enzyme such as Xho1 and BamH1 that cut my final construct into 3 and 4 segments respectively.
Thanks
Hi Nasrin.
GFP expression alone would not tell whether mutation occurred in non-coding sequences in the vectors or the colony picked is mixed colonies of transformants. Reduce the bacteria concentration when you spread. Pick several colonies separately as early as possible, and repeat the above procedure again to pick a single cell-derived colony.
Regards.
Dear Mr. Lee
Thanks for your answer.
After doing PCR, I sent my inserts to be sequenced.
In addition, restriction analysis for these 2 inserts happened in an acceptable pattern, before insertion of GFP.
I think streak culturing might be helpful for me. But I am searching for other possibilities.
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