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molecular biology

Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.

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molecular biology

Postby biotechbuddy » Fri Aug 27, 2010 2:39 pm

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) :?:
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Postby JackBean » Fri Aug 27, 2010 4:41 pm

question is, whether is it really intron or just some inserted piece of DNA? How did you get your sequence? From gDNA or mRNA?
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
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Re:

Postby biotechbuddy » Mon Aug 30, 2010 6:32 am

JackBean wrote: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. :?:
Last edited by biotechbuddy on Mon Aug 30, 2010 8:06 am, edited 1 time in total.
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Postby biotechbuddy » Mon Aug 30, 2010 6:35 am

Thankz allot Sir for your reply.
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Postby JackBean » Mon Aug 30, 2010 9:13 am

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 ;)
http://www.biolib.cz/en/main/

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Postby biotechbuddy » Mon Aug 30, 2010 10:00 am

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.
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Postby JackBean » Tue Aug 31, 2010 3:18 pm

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

It migth work, but doesn't have to.
http://www.biolib.cz/en/main/

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