Cloning in frame into pcDNA 3.1(+) vector - a quick question (Mar/30/2008 )
The vector map from invitrogen does not show any translation frame in the vector. Can I clone my insert at any restriction site at MCS? Does that mean that my insert will be translated in frame, provided that there is an ATG codon in the beginning?
Never done any cloning into mammalian expression vectors, so not sure...
-molecular dummy-
QUOTE (molecular dummy @ Mar 30 2008, 07:29 PM)
The vector map from invitrogen does not show any translation frame in the vector. Can I clone my insert at any restriction site at MCS? Does that mean that my insert will be translated in frame, provided that there is an ATG codon in the beginning?
Never done any cloning into mammalian expression vectors, so not sure...
Never done any cloning into mammalian expression vectors, so not sure...
No, you need to look at the sequence to find a site that will be in frame with your insert. If your vector map does not show the frame, then download the sequence, do a digest using a computer program to line up your MCS, ATG, and anything else of interest in the vector. Then choose which site to use.
-smu2-
Well in pcDNA 3.1(+ or -), if you read the manual they tell you that you need to have your own start codon (ATG) and kozak squence which means translation will start from your ATG. One thing though is that you need to make sure you don't introduce any other ATG before your ATG in the MCS.
I worked with pcDNA3.1 a lot. So, I think if you have ATG in your insert thats gonna be fine
Good luck
-anwar_mt-
QUOTE (anwar_mt @ Mar 31 2008, 08:44 PM)
Well in pcDNA 3.1(+ or -), if you read the manual they tell you that you need to have your own start codon (ATG) and kozak squence which means translation will start from your ATG. One thing though is that you need to make sure you don't introduce any other ATG before your ATG in the MCS.
I worked with pcDNA3.1 a lot. So, I think if you have ATG in your insert thats gonna be fine
Good luck
I worked with pcDNA3.1 a lot. So, I think if you have ATG in your insert thats gonna be fine
Good luck
Well I agree with anwar. there is a plenty of confusion among the new comers as to what actually is "in-frame". The concept of in-frame only implies when we are trying to clone our sequence into a plasmid which contains either some translation regulatory element, like kozak sequence or ATG etc etc or in other cases an N-terminal or C-terminal tag so that when we clone our sequnce into that plasmid, the ORF which will be subsequently translated should not suffer from the frame shift. but when we have a complete ORF provided by ourselves, to the vector, and the vector doesnot contain any sequnces which will be translated (ATG, Kozak, N-terminal or C-terminal tags) along with our insert, bringing in-frame isnt an issue at all. you can simply select any restriction site and just clone your sequence in it.
-Muhammad Umer-
QUOTE (smu2 @ Mar 31 2008, 07:23 AM)
QUOTE (molecular dummy @ Mar 30 2008, 07:29 PM)
The vector map from invitrogen does not show any translation frame in the vector. Can I clone my insert at any restriction site at MCS? Does that mean that my insert will be translated in frame, provided that there is an ATG codon in the beginning?
Never done any cloning into mammalian expression vectors, so not sure...
Never done any cloning into mammalian expression vectors, so not sure...
No, you need to look at the sequence to find a site that will be in frame with your insert. If your vector map does not show the frame, then download the sequence, do a digest using a computer program to line up your MCS, ATG, and anything else of interest in the vector. Then choose which site to use.
i guess that I ought to edit my post, as I didn't read the title carefully enough. I didn't realize that the poster was referring to a specific clone - thought they were referring to something else. As such, take the advice of subsequent posts. Guess I need some more zzzzzz... too sleepy.
-smu2-
QUOTE (smu2 @ Apr 1 2008, 02:12 AM)
QUOTE (smu2 @ Mar 31 2008, 07:23 AM)
QUOTE (molecular dummy @ Mar 30 2008, 07:29 PM)
The vector map from invitrogen does not show any translation frame in the vector. Can I clone my insert at any restriction site at MCS? Does that mean that my insert will be translated in frame, provided that there is an ATG codon in the beginning?
Never done any cloning into mammalian expression vectors, so not sure...
Never done any cloning into mammalian expression vectors, so not sure...
No, you need to look at the sequence to find a site that will be in frame with your insert. If your vector map does not show the frame, then download the sequence, do a digest using a computer program to line up your MCS, ATG, and anything else of interest in the vector. Then choose which site to use.
i guess that I ought to edit my post, as I didn't read the title carefully enough. I didn't realize that the poster was referring to a specific clone - thought they were referring to something else. As such, take the advice of subsequent posts. Guess I need some more zzzzzz... too sleepy.
I would like to add a question to molecular dummy's query. If our vector for eg, has 68 nucleotides before the ATG, meaning A(nucleotide 69), T(nucleotide 70) and G (nucleotide 71), would the translated sequence still be in frame or there would need to be 69 (multiples of 3) before the ATG before it can translate correctly?
-elsh-
The frame is established by the ATG start codon. The number of nucleotides in the 5' UTR of the transcript is not relevant in determining the frame.
-phage434-