Hi
I am having issue's with Gibson assembly, seeing as I am new in the filed of cloning, expert feedback would be very much appreciated
I am trying to clone two fragments (frag 1 [185bp, 145bp w/o overhangs]) and Frag 2 [224bps, 192bp w/o overhangs]) into a7.6kb plasmid via Gibson assembly.
1) My first issue is with PCR amplification of my two fragments (1, 2), using primers that add overhangs (22 & 18 bp over hangs to frag 1 and 10 & 22 bp overhangs to frag 2). The issue is that during PCR I seem to get non-specific bands across all annealing temperatures for both my fragments. Whats more interesting is that around the anticipated size range for my fragments (185bp and 224) there appears to be duplicate bands within close proximity (labelled in attached power point slide 1 & 2).
2) I did go ahead to purify Frag 1 and 2 from the gel by cutting out the duplicate bands at the anticipated size, following Gibson reaction I tried confirming successful ligation of the insert into the vector by designing primers that amplify the vector backbone from a region upstream of the insert integration site to a region downstream of the insert integration site (such that amplicon size without insert is 311 bps and with insert is 691 bps). Following Gibson assembly reaction I was unable to detect any amplicons of 691bps (slide 3 attached power point) suggesting my Frag 1 & 2 did not integrate into the plasmid. I did go ahead to transform TOP10 with this same Gibson reaction sample, I got colonies, however they were also negative for the insert following colony PCR.
I would like any suggestions to improve the quality of my insert fragments amplified by PCR and my likely hood of success in subsequent Gibson reactions.
Thanks
Ifeanyi
1. Fragment amplification problem
- Try to check if your primer or overhang will bind to other parts of the vector or inserts.
- when determining your annealing temp, you should just use your primer without the overhang because when you are doing amplification the part that anneals is the part that is complementary to the insert, not the overhang part. Eg., your primer is 20 nucleotides plus 22 nucleotides of overhang. So when you are calculating the melting temp (you can use any online melting temp calculator), just use the 20 nucleotide part of the primer.
2. Cloning problem
- do you do any positive control? Gibson provides positive control in their kit, it will be better if you can try out the positive control and see if it works to ensure that the cloning kit protocol is ok.
- try to use 1:3 or more molar ration for your vector: insert
3. Alternative method
- If you are looking for alternative, I'll recommend Hot-Fusion method. I've used both Hot-Fusion and Gibson before and I can assure you that Hot-Fusion method efficiency and success rate are comparable to Gibson.
Since both of the fragments are ~200bp, just go ahead to synthesize. do not need to struggle with the PCR.
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