5 replies to this topic

[SF_HK]

Hi,

It took 2 months to clone my insert in pcdna3.1(-)/myc/his (invirogen) vector. However, upon sequening I realise a problem.

I wanted to tag my full length insert and thus did not include the stop codon. Upon sequencing I find that he forward sequence is in frame but I have an additional nucleotide at the end of my sequence? so:

cdna sequence: 5;attTAG (TAG= stop codon)

my intended insert seg: 5'-att only followed by the vector sequence

my wrong insert seq: 5'-attT-followed by the vector sequence (so basically, I know have the additional T from the TAG that I intended to delete)

So, I obviously won't be geting my tag but can I detect my protein using the protein antibody? Or wil my protein be affected?

Thanks

[almost a doctor]

SF_HK, on May 21 2009, 04:23 PM, said:

Hi,

It took 2 months to clone my insert in pcdna3.1(-)/myc/his (invirogen) vector. However, upon sequening I realise a problem.

I wanted to tag my full length insert and thus did not include the stop codon. Upon sequencing I find that he forward sequence is in frame but I have an additional nucleotide at the end of my sequence? so:

cdna sequence: 5;attTAG (TAG= stop codon)

my intended insert seg: 5'-att only followed by the vector sequence

my wrong insert seq: 5'-attT-followed by the vector sequence (so basically, I know have the additional T from the TAG that I intended to delete)

So, I obviously won't be geting my tag but can I detect my protein using the protein antibody? Or wil my protein be affected?

Thanks

I believe that if you dont have a stop codon your protein will be "tagged" with whatever the sequence becomes with this new T until the next stop codon.  

I know is not good news, but I'll re-clone it.      

I might be wrong thou, so more advice here will be welcome.  

[phage434]

I agree.  Presumably your second try at cloning will be a lot quicker now that you know how to do it.

[swanny]

You can also try something like Quick Change mutagenesis kit from Stratagene.

[SF_HK]

I tried the mutagenesis kit form Strtagene. There were 20 colonies picked two and the extra nuleotide wasn't deleted.  Will pick all the 20. I have a feeling doing a deletion is more complicated than a mutation. I did a single base pair mutation last week and got 100% efficiency. All my clones had the mutation.

[HomeBrew]

Why did it take two months to clone your insert?