Its a real simple question, but i have no biotech base, and no one in my lab is free to provide me guidance. That's why i am asking this question here.
I have been trying cloning a gene for almost 1 year, and its a total mess because the size is quite large (2340bp CDS). I was using T-vector, since other vectors were not working, so I have to clone it to T-vector first and then use that plasmid to clone it to pSuper1300 vector with GFP tag. Now i have found one positive colony, and the sequencing results from the company is also ok. The alignment also seems alright. But my professor said thats not the way to align and confirm a gene. First you should translate it to the protein form, and then blast that protein on NCBI to confirm.
Now which kind of blast should i use? I used different blast types, N_N, N_P, and P_N have more than 99% score, but P_P has 29% score, however each kind of blast matched the same kind of gene or protein. But I am not sure which one is the correct method of finding it.
Please give your valuable opinion. If you don't understand my question, let me know kindly (as my knowledge is very poor, and I even don't know the correct way of asking questions in molecular biology)..
Thanks in advance
By western blot.
However, sequence analyses should be optimally done by NGS. Usinf databases I would propose to use a blastp or blastx algorithm.
Andreas Eisenreich Sir do you mean I should combine the sequences (sent by the sequencing company) and then translate it to protein, and then run BLASTp or BLASTx, right?
No...you can a) perform NGS (e.g. exon expression analyses) or isolate your protein of inerest and perform LC/GC-MS/MS to identify it.
Annother approach would be a simple Western blot with a specific antibody (or Performance of an ELISA).
Yes sir, thats what my Professor told me, after analyzing the sequences on a software, i will do a western blot. But before that, I need to translate the sequences with the help of a software, to make sure it makes a protein without any end codons.
Ok, you need the AA sequence (maybe you can get it - as mentioned by you - from translation of the cDNA sequence to AA sequence, or more reliably e.g. via LC-MS/MS).
You take the Sanger sequence data and run a BlastX. That will tell you the possible proteins that match your sequence.
Or you can put your Sanger data into a translation algorithm, look at all 6 reading frames, and see if it's an exact match to the known amino acid sequence of your protein.
After i got sequences of the clone from the sequencing company, i removed the flanking regions at both ends (peaks that are ambigous).. then i allinged all the sequences together to see where they overlap. Hence i removed the overlapping regions, and made one full sequence, then translated it to protein form... Now when i BLAST that protein sequence, the result shows the same protein which i am concerned about... But when i showed these results to the professor, he said the protein frames have too much end codons (shown as stars), so it shows your clone is not a reliable one, and the reason might be a bad cDNA...
So after 1 year of trying, i found one clone, which is not reliable...
So you people suggest me, what should i do? keep trying or just quit?
Muhammad Owais Shahid I'd try again. 2340 bp isn't that large of an insert to clone. Make fresh cDNA or get some from a colleague with good lab skills, design your primers carefully, and start over. I've had great luck with the TA Topo cloning kit. It's a bit expensive, but it works so well it's a good investment.
Good luck!
Dear Muhammad
I hope these papers will hlep you to get your answer
Article Cloning, Mapping, and in vitro Transcription--Translation of...
Article A general method for maximizing the expression of a cloned gene
Regards
i figured out how to confirm it. I want to share my experience here so that others can helped:
1) Ask the sequencing company to sequence the full length of the gene in bacterium.. Better to design a primer after every 800bp and send it to the company along with the transformed bacterium.
2) After receiving sequence files, remove the flanking regions from ends of the fragments. It can be done easily with CHROMAS software
3) Using DNAman, load all the sequences along with your original gene sequence (from the Database) and do 'Sequence assembly' and select 'overlap' to see where they are overlapping, and get the consensus sequence. You can see where there are differences in the sequences. Consensus gives the most suitable bases where there is a difference.
4) Translate the consensus sequence and see which longest protein it is making Using 'Translation overview' and 'Translation' in DNAman)
5) Align it with the original protein of the gene (from the Database). It should be highly similar. If some of the protein bases are not similar, it might or might not be ok, because some SNPs are acceptable but some are not.
6) With western blot or other experiments, confirm it again
Hope it will help people
Regards
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