I had a BL21 (DE3) cell which I always use to express GFP protein which fused to a coil. Based on the vector map given to me, this gene is inside the pQE80 vector which has resistance to ampicillin. I induce its expression by IPTG and it expresses a protein with 35 Kda MW (expected data based on desired protein MW). I should mention the colonies which i get after transformation also have green color! I used this construct to do RF cloning and after sequencing, i realized the sequence doesn't have homology neither to a template nor desired construct after cloning!
I got suspicious about my template stock and did streak culture from BL21 stock and pick a single colony and after pls prep and sending sequencing, it doesn't show any alignment with a vector map I have! I designed primers from the middle of GFP and sequencing result with them also doesn't show any homology!
It means I express a protein from nowhere! I should tell also this coil interacts with another coil and I prove their interaction to each other. so coil construct and GFP sequence are both there but sequencing doesn't give me any data! sequencing did with the pQE forward and reverse universal primers, pET universal primer, T7 promotor, T7 terminator, and designed primers from inside of GFP! all fail!
If there is contamination, after picking a single colony I still have it! there is doubt there are more than the desired construct inside the cell which sequencer can't read it?! how can I get my desired construct?
I totally get confused and I will be so thankful for any suggestion.
Is the sequence data you are evaluating just the text file or do you have access to the raw files?
dear Wolfgang Schechinger Thanks for your reply:) I have the sequencing results as pdf and ab1 files. The result of sequencing in the pdf file shows no response! after aligning raw data (ab1 file) in the genome compiler with my desired vector, it doesn't show any homology!
Did you try to transform XL1 with your plasmid? XL1 amplifies the plasmid without introducing it into its genome. You can get plasmids of higher purity from them, maybe that improves your sequence data.
Or sometimes it is just such an easy thing like making your sequence data reverse complement...
It is hard to understand what went wrong. Obviously, your plasmid expresses GFP and at some point you mention that primers from the middle of GFP result in sequence that show no homology. If you get real sequencing results from GFP primers, you should at least start with GFP sequence. If you do this in both direction you should get enough information about the fusion and the vector.
Always try to BLAST your unknown sequences with the NCBI NR database.
If nothing works, there must have been a total mixup at some point.
If this is the case, try to get the original plasmid from the creator. Be aware that you have to use mutated monomeric GFP. The original GFP will dimerize and therefore interfere with your assay.
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