Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

in frame problem after 3 months of cloning!


  • Please log in to reply
5 replies to this topic

#1 SF_HK

SF_HK

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 195 posts
2
Neutral

Posted 21 May 2009 - 07:23 AM

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

#2 almost a doctor

almost a doctor

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 244 posts
15
Good

Posted 21 May 2009 - 07:59 AM

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. :)

#3 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,476 posts
251
Excellent

Posted 21 May 2009 - 01:37 PM

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

#4 swanny

swanny

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 367 posts
8
Neutral

Posted 21 May 2009 - 06:07 PM

You can also try something like Quick Change mutagenesis kit from Stratagene.
Heart disease kills more women than breast cancer, but heart attack symptoms differ from men's symptoms. Get to know your heart... it could save your life.

#5 SF_HK

SF_HK

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 195 posts
2
Neutral

Posted 21 May 2009 - 07:34 PM

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.

#6 HomeBrew

HomeBrew

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 930 posts
15
Good

Posted 22 May 2009 - 02:46 AM

Why did it take two months to clone your insert?




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.