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Last time when I cloned a short gene which is about 1kb everything was completely fine the sequence was exactly the same as I wanted it to be, however as soon as I go for a bigger insertions I immediately get nucleotide polymorphisms. So what is the common strategy to overcome this problem? Thanks for advices in advance!
I am already getting crazy with this mutagenisis... I see that most of you are using kit from Stratagene, however I decided to order one from Invitrogen - Geneart seria.
I desinged my primers to get mutated site in the middle... the Tm is about 80, number of base pairs - 41.
Tried already several conditions for PCR: incresead - decreased the amount of template from 20 to 100ng, changed annealing temperature from 55 to 62, used different amount of polymerase 4-20 Units, added DMSO to prevent dimers... nothing helped... no idea what to do next...
I read somewhere that primers should be HPLC cleaned then PCR works better, could it be the case?
the most intriguing thing is when I load gel with PCR products I always load one more well with the same amount of template as a kind of positive control and the thing is that I see band on around 5kb only in the case of positive control... what happens with the template which I use for PCR? why I dont see it on the gel if the amount is the same?
Any ideas what to try next?
Thanks in advance!
Maybe you can find someone who has the full length ORF in some plasmid, and is willing to provide you with? Most people are, if you acknowledge them (and cite).
I am from Australia. It costs about 1500 dollars to get a shipment on dry ice from US, so that's a bit complicated.
In a case if I want to check collocalisation of my overexpressed protein tagged by GFP should I knockdown endogenous protein? or it is not neccecary since the amount of overexpressed protein is usually 5-10 times higher? does it make sence to reduce amount of overexpressed protein in order to make it more comparable with endogenous one?
If your cell line expresses this protein, then you should knock it down first, to have a clearer effect of the mutation. So your approach is quite good.
good point, I have to check
How about conservation of this particular residue (of your point mutation)?
You are right, we can buy human cDNA basicaly it's just a matter of money...
Human cell line which we use express its own endogeneous protein. I thought of transfecting the cell line with siRNA to reduce the competition with our overexpressed protein that would also an explanation for reveiwers why we use protein from another species (to supress only endogenous protein but not the one we overexpress). What do you say is a right direction to follow?
P.S. protein is highly conserved.
I have a question regarding the overexpression studies. We have identified a new de novo mutation in a human and would like to perform some functional analysis using cell lines. We think that it's better to use human cell lines since the mutation observed in humans, however, we would like to overexpress mouse construct tagged with GFP or whatever else ... (one of the reasons is that we don't have suitable human cDNA to clone the construct). So the question is would it be the case for reviewers to pay attention on this fact? How reasonable to overexpress proteins from other species?
I saw quite a lot of papers where people did it in this way and since they got published they apparently had no so many difficulties...
The homology between human and mouse proteins more than 90%, the protein indirectly regulates calcium signalling.
I am a bit confused since my new project requires to perform a cloning of a huge human gene consisting of 31 exons, all together they are about 8 kb BUT!!! The problem is that exons are separated by introns which are more than 10kb each. Therefore, the only possibility I see right now is to subclone all of 31 exons individually and afterwards ligate them which might be extremely time consuming procedure... Would it be an another possibility to get full length cDNA of this gene? currently I have only the genomic DNA from this sample...
Thanks in advance for your help!