Hi everyone. I am attempting to amplify a target gene for use in Gibson assembly and have been getting the incorrect band size on my gel.
Initially I was having problems getting the target gene to amplify correctly, so my supervisor suggested designing primers to amplify the whole gene (for Gibson assembly we designed the primers to "cut" the target gene before the stop codon in order to attach it to a GFP tag). This worked very well and I got the correct band size there (6kb). I purified the product from this PCR using the QIAquick PCR Purification Kit and used this as the template to amplify the target gene with the Gibson assembly primers.
But now that I am trying to amplify the target gene with the Gibson assembly primers and am getting the wrong band size on the gel (I am seeing one band of ~1kb). I have tried using a different enzyme (the first time around I used Longamp and I also tried it with Taq DNA Polymerase), but that didn't work. My thought is that there is something wrong with my primers, but wondered if anyone has any other ideas.
This overall process has worked with the other genes I am working on
Kethry,
What is the overlap size of the primers are you using? I usually create primers with overlaps between 25-30 bp. To amplify large fragments I use Taq Platinum (adding KB extender in the reaction). Have you ever tried the NEB builder Assembly tool? ( https://nebuilder.neb.com/#!/ ). This tool can help you by creating the ideal primers for your construction.
Hi Kethry,
The problem you were facing while you tried to amplify a targeted segment from original template seem to persist while you try to amply the same region from purified PCR product of whole gene.
I have a solution which actually worked in similar situation. As you have amplified 6kb fragment (along with stop codon you were trying to avoid), you can now use the whole gene (along with stop codon) for your Gibson assembly. Only trick you have to do is design the primer for your backbone with overlap (of 20-25bp) without the stop codon. Once you amplify the backbone, you can carry out Gibson assembly reaction with your whole gene insert and still get the fragment you were trying to amplify into the backbone.
The original Gibson assembly process splits the overlap (20bp) into two halves (10 bp in the primers used to amplify the insert and other 10 bp in the primers used to amplify the backbone). However, for situations like this, you can use longer overlap (whole 20bp) in the primers used to amplify the backbone and proceed with the assembly reaction. I suggest to screen 7 or 8 clones through sequencing before you proceed to the next experiment with your construct.
Best wishes for your research.
Thanks for the responses. The overlap on the primers is around 20bp. I have used the NEBuilder tool before, but did not think to use it for this.
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