molecular biology

[biotechbuddy]

can u please help me regarding this, i have cloned my gene (800 bp long) but when I sequenced it I found some 50bp of introns into it can I transfect this to melanoma cell line to get protein expression.
Vector used EGFP Nl.
DNA :CD82
protein: a tetraspanin (CD82)

[JackBean]

question is, whether is it really intron or just some inserted piece of DNA? How did you get your sequence? From gDNA or mRNA?

[biotechbuddy]

question is, whether is it really intron or just some inserted piece of DNA? How did you get your sequence? From gDNA or mRNA?

I got this by mRNA & it is an intron sequence of Same gene(CD82) i confirmed it by BLAST.
Also,If I will transfect this vector (EGFP Nl with CD82 insert) in B16bL6 melanoma cell line , then that will get into nucleus or will remain into cytoplasm.

[biotechbuddy]

Thankz allot Sir for your reply.

[JackBean]

I have never worked with mammalian cells, but I think there are used systems for incorporation into genomic DNA.

Is it whole intron or just part of it? Maybe you have discovered alternative splicing

[biotechbuddy]

there are 6 introns in all , out of which 5 introns got removed but one complete intron was not removed, which is matter of concern to me. accordingly after transfection it should get removed , but our team is not sure about it.

[JackBean]

Well, that could really be some alternative splicing, you should check it

It migth work, but doesn't have to.