Deletion Constructs w/Quik Change 2 Kit problemos - no colonies forming 14aa consecutive deletion (Jul/05/2005 )
I am in the process of trying to use the Quik Change II kit from Stratagene (have used it for insertion of stop codons).
This time however I am making a 14amino acid (consecutive aa's) stretch using this kit. Larger deletions have been done w/this kit, how I am unsure.
I am not getting colonies after the transformation.
I will supply any info. that anyone wants to know.
BTW, my primers are GC rich, they are 70%. My extension time is 5mins, 68C, for 18 cycles.
I have used this exact construct (including insert) for the stop codon mutation.
Hi viper,
I haven't attempted a deletion that large using Quickchange, however, when I had to delete 10 consecutive a.a. what I did was a design a reverse primer immediately uptream of the deletion and a sense primer immediately downstream of the deletion. Carry out a standard PCR using Pfu (or any proof reader leaving absolute blunt ends) amplifying the entire plasmid, phosphorylate, and ligate. This has a number of advantages including smaller primers (less secondary structure), checkpoints along the way, and in my opinion more chance of working.
Hope this helps,
Scott
I haven't attempted a deletion that large using Quickchange, however, when I had to delete 10 consecutive a.a. what I did was a design a reverse primer immediately uptream of the deletion and a sense primer immediately downstream of the deletion. Carry out a standard PCR using Pfu (or any proof reader leaving absolute blunt ends) amplifying the entire plasmid, phosphorylate, and ligate. This has a number of advantages including smaller primers (less secondary structure), checkpoints along the way, and in my opinion more chance of working.
Hope this helps,
Scott
Thanks, this is very similiar Stratagene's Excite Kit that I am going to try. It uses PO4'd primers and basically does the same thing including the need for the ligation.
THANKS
I haven't attempted a deletion that large using Quickchange, however, when I had to delete 10 consecutive a.a. what I did was a design a reverse primer immediately uptream of the deletion and a sense primer immediately downstream of the deletion. Carry out a standard PCR using Pfu (or any proof reader leaving absolute blunt ends) amplifying the entire plasmid, phosphorylate, and ligate. This has a number of advantages including smaller primers (less secondary structure), checkpoints along the way, and in my opinion more chance of working.
Hope this helps,
Scott
Hi viper,
I am trying to do the same experiement trying to delet 14 AAs and our lab cannot use any kit will appreciate if you can send detailed strategy how and whcih region for primer design and how PCR performed. Many thanks.
I haven't attempted a deletion that large using Quickchange, however, when I had to delete 10 consecutive a.a. what I did was a design a reverse primer immediately uptream of the deletion and a sense primer immediately downstream of the deletion. Carry out a standard PCR using Pfu (or any proof reader leaving absolute blunt ends) amplifying the entire plasmid, phosphorylate, and ligate. This has a number of advantages including smaller primers (less secondary structure), checkpoints along the way, and in my opinion more chance of working.
Hope this helps,
Scott
Hi viper,
I am trying to do the same experiement trying to delet 14 AAs and our lab cannot use any kit will appreciate if you can send detailed strategy how and whcih region for primer design and how PCR performed. Many thanks.
Scott I am doing what you mentioned...it's Stratagene's Excite Kit actually...However my primer's appear to be unable to PCR at all. Out of 24mer primer size, I have 18 base pairs that are G/C....This is proving very difficult.
I have tried annealing temps of 60, 65 and 70. The 70C reactions had4% DMSO as well.
All I get are 250bp/300bp products....
If I cannot use PCR to make these mutations...what can I do? I wish Stratagene made their Chameleon Kit, because that kit could do it. Every company thinks PCR based methods are the best..and they aren't.