Sequence error in cloned expressed gene - how to correct it? - (Jul/24/2006 )
QUOTE (HomeBrew @ Jul 26 2006, 09:54 PM)
I agree with wbla3335 -- if you do have a single amino acid mutation, there's no evidence to support that NCBI's sequence is "correct" and yours is "wrong". How many entries are there for this gene in GenBank? Are the from different labs? Are they all exactly the same? Are you using the exact same genus/species/strain of whatever organism this is (what organism is it, BTW) as was used to generate the sequence in the database?
Why did it take 3 months to clone the gene? I'm not being critical, it's just that if I found myself in the same situation, I'd just re-clone the gene rather than try a mutagenesis procedure or kit, but I'd expect recloning to take more like three days rather than three months.
Also, I do not fully understand what happened here. Can you re-explain this:
The reverse (complement) of CCA is TGG, not ACC. Why are you "reading from the end of the gene"?
Why did it take 3 months to clone the gene? I'm not being critical, it's just that if I found myself in the same situation, I'd just re-clone the gene rather than try a mutagenesis procedure or kit, but I'd expect recloning to take more like three days rather than three months.
Also, I do not fully understand what happened here. Can you re-explain this:
QUOTE
Previously when I checked the sequences after sequencing result, I happily check and didnt realise that since I used blast from NCBI, the Reverse reaction starts from the end of the gene.And unfortunaltely, there is a mismatch at the end of the gene reading from the end of the gene, I get CCA when it was supposed to be CCG. After I translated it, the amino acid remains the same. BUt as I hav realised, i was supposed to read it the other way round, meaning now the mismatch is from GCC to ACC which have two totally diferent amino acid!!!!!!!!!!!
The reverse (complement) of CCA is TGG, not ACC. Why are you "reading from the end of the gene"?
Oh, it is a human gene. Well, thats why I started by feeling so stoopid! its because when I blast the gene with the clone, the clast result starts from the end of the gene, and stoopid me go and read from there onwards. Haiz
Erm, i agree its very looooooooong to clone for 3 months, cos I started with PCR then cloning it. And the problem is I am studying and doing my project at the same time so it kinda made my progress slower cos can only do a few days a week
-Fossil-
QUOTE (T. reesei @ Jul 26 2006, 09:56 PM)
in that case mutation may be solve of your problem
may be u can try with 1 sapmle........ur original one (with mismatch)
and mutated one
best of luck
may be u can try with 1 sapmle........ur original one (with mismatch)
and mutated one
best of luck
I shall check out if tats possible and if its within the budget for the project. Thanks anyway!
-Fossil-
anyway plz let us inform your final result. that is after end of your project what result you will get
-T. reesei-
QUOTE (T. reesei @ Jul 26 2006, 10:09 PM)
anyway plz let us inform your final result. that is after end of your project what result you will get
Yup, I will thank you so much!! well, I think before tat, I will have more questions for u guys haha
-Fossil-
Can you analyze your sequence in some kind of protein structure prediction program to see whether it would fold the way you expect it to?
Or you can express it somewhere and test the function and if it does what it is supposed to then the structure is ok.
Do you know which epitope your 3`antibody recognizes?
-chalet2-
QUOTE (chalet2 @ Jul 27 2006, 09:46 PM)
Can you analyze your sequence in some kind of protein structure prediction program to see whether it would fold the way you expect it to?
Or you can express it somewhere and test the function and if it does what it is supposed to then the structure is ok.
Do you know which epitope your 3`antibody recognizes?
Or you can express it somewhere and test the function and if it does what it is supposed to then the structure is ok.
Do you know which epitope your 3`antibody recognizes?
Hi chalet2,
I couldnt find such a programme tat is capable of telling me how the protein is going to fold. Regarding wat epitope that my antibody will bind, I am not sure either cos I searched the Ab tat I am buying, it only mentioned the immunogen is at either the N or C terminal.
-Fossil-