Ugrent help in oligo insertion - (Sep/23/2009 )
Dear mates,
I have pcDNA3.1 plasmid (Invitrogen), that contains aminigene construct.
In this minigene sequence, i have a motif that i want to exchange with a 70 pb coat protein binding sequence that form a loop 2ndery structure, so it is sort of mutagenesis.
The thing that i started with is to design a sense and anti sense primers that contains a 15 bp minigene specific sequence and a 35 bps tail on each primer for the coat protein(50 bp each primer), and tried to amplify the whole plasmid with PFU enzyme.
Unfortunately i didnt get any results( i ran the PCR with 15 cycles and around 300ng of template, elongation step for 5,3 min. The total size of the plasmid is around 5.5kb).
When i Discussed it withmy superviser, i suggested that i can order the whole insertion(70BP) as an oligo from the primers company and cloning it.
The cloning that i thought about for this step is to amplify the whole plasmid head to head primers that contains half of a specific sticky ends restriction sites that will exist in the oligos aswell and perform the cloning afterward.
What he said is are you crazyy!!!!!!!!
You will never git a 7o bp oligo from a company with out a mutation so, you will be serquencing and looking for the right clone for the rest of your life.
So please any body can suggest any method or an idea to help me with this????
Thanks in advance
thegene on Sep 23 2009, 02:12 AM said:
I have pcDNA3.1 plasmid (Invitrogen), that contains aminigene construct.
In this minigene sequence, i have a motif that i want to exchange with a 70 pb coat protein binding sequence that form a loop 2ndery structure, so it is sort of mutagenesis.
The thing that i started with is to design a sense and anti sense primers that contains a 15 bp minigene specific sequence and a 35 bps tail on each primer for the coat protein(50 bp each primer), and tried to amplify the whole plasmid with PFU enzyme.
Unfortunately i didnt get any results( i ran the PCR with 15 cycles and around 300ng of template, elongation step for 5,3 min. The total size of the plasmid is around 5.5kb).
When i Discussed it withmy superviser, i suggested that i can order the whole insertion(70BP) as an oligo from the primers company and cloning it.
The cloning that i thought about for this step is to amplify the whole plasmid head to head primers that contains half of a specific sticky ends restriction sites that will exist in the oligos aswell and perform the cloning afterward.
What he said is are you crazyy!!!!!!!!
You will never git a 7o bp oligo from a company with out a mutation so, you will be serquencing and looking for the right clone for the rest of your life.
So please any body can suggest any method or an idea to help me with this????
Thanks in advance
Please if it is not clear i can explain more::::
Thanks
thegene on Sep 23 2009, 02:12 AM said:
I have pcDNA3.1 plasmid (Invitrogen), that contains aminigene construct.
In this minigene sequence, i have a motif that i want to exchange with a 70 pb coat protein binding sequence that form a loop 2ndery structure, so it is sort of mutagenesis.
The thing that i started with is to design a sense and anti sense primers that contains a 15 bp minigene specific sequence and a 35 bps tail on each primer for the coat protein(50 bp each primer), and tried to amplify the whole plasmid with PFU enzyme.
Unfortunately i didnt get any results( i ran the PCR with 15 cycles and around 300ng of template, elongation step for 5,3 min. The total size of the plasmid is around 5.5kb).
When i Discussed it withmy superviser, i suggested that i can order the whole insertion(70BP) as an oligo from the primers company and cloning it.
The cloning that i thought about for this step is to amplify the whole plasmid head to head primers that contains half of a specific sticky ends restriction sites that will exist in the oligos aswell and perform the cloning afterward.
What he said is are you crazyy!!!!!!!!
You will never git a 7o bp oligo from a company with out a mutation so, you will be serquencing and looking for the right clone for the rest of your life.
So please any body can suggest any method or an idea to help me with this????
Thanks in advance
thegene on Sep 23 2009, 06:27 AM said:
thegene on Sep 23 2009, 02:12 AM said:
I have pcDNA3.1 plasmid (Invitrogen), that contains aminigene construct.
In this minigene sequence, i have a motif that i want to exchange with a 70 pb coat protein binding sequence that form a loop 2ndery structure, so it is sort of mutagenesis.
The thing that i started with is to design a sense and anti sense primers that contains a 15 bp minigene specific sequence and a 35 bps tail on each primer for the coat protein(50 bp each primer), and tried to amplify the whole plasmid with PFU enzyme.
Unfortunately i didnt get any results( i ran the PCR with 15 cycles and around 300ng of template, elongation step for 5,3 min. The total size of the plasmid is around 5.5kb).
When i Discussed it withmy superviser, i suggested that i can order the whole insertion(70BP) as an oligo from the primers company and cloning it.
The cloning that i thought about for this step is to amplify the whole plasmid head to head primers that contains half of a specific sticky ends restriction sites that will exist in the oligos aswell and perform the cloning afterward.
What he said is are you crazyy!!!!!!!!
You will never git a 7o bp oligo from a company with out a mutation so, you will be serquencing and looking for the right clone for the rest of your life.
So please any body can suggest any method or an idea to help me with this????
Thanks in advance