Hello,
I have been working with Gibson Assembly in order to create three separate plasmids. I have ran PCR on pET28a+ and an already cloned plasmid containing two genes of interest. The GC content and primer Tm are normal (within 40-60% and 58-68 degrees for Q5 High Fidelity Polymerase PCR respectively). I am confident the PCRs have worked as gel electrophoresis and sequencing has verified. The backbones are 5-7 kilobases in length while the inserts range from .7-2 kilobases. The backbones were PCRed following the NEB protocol and using the NEB online Tm Calculator. The inserts were created with the same protocols, but the primers have overhangs between 20-45 bp in length. No Gibson's have worked thus far when using 1:1 equimolar ratios nor 1:2 backbone : insert ratios when using the NEB Bio Calculator. All Gibson Assembly reactions were ran in the thermocycler at 50 degrees celsius for 15 minutes.
The primary goal for one of the plasmids is to simply take out the CMR encoding gene and reinsert it such that the reverse complementary nucleotide sequence is present. This so that chloramphenicol resistance can not be expressed off the template DNA. Only when read in the 5' -> 3' direction should CMR be produced. This is essential for future experiments.
I am trying to do the same for another plasmid construct, except I would like to remove an additional gene encoding for an RNA polymerase while reinserting CMR in the same fashion.
Finally for the third construct I would like to insert a 2kb insert into my pET28a+ backbone.
None have worked thus far. Sequencing has shown that only the backbone DNA is present and no DNA was ever inserted. I transformed my Gibson Assembly products into DH5alpha cells and plated following manufacture's instructions.
Any ideas on how to troubleshoot?
increase the incubation time to 1 hour and switch to neb-10-beta (I have had bad results with dh5 alpha) and make sure you backbone is properly gel purified.
Do your backbone clones lack the CMR?
If yes, are the ends you have generated just by chance prone to work for Gibson assembly?
If not, ( I guess you ruled that out) you have a problem with the parental plasmid. For the third construct you may consider using a conventional vector cut with two enzymes in the MCS.
Draven Rane
make sure that your PCR products are of correct sizes and gel purify everything, vectors too. Don't rely on DpnI too much, this is bad enzyme. Wash DNA pellets with 70% ethanol.
Using other cells than DH5alpha might help too.
Obvious question, but did you preform a DPN digest on your plasmid backbone?
Aleksey Karpitskiy Oskar Laur I did gel excisions and purified for all backbones and inserts. I am attempting using DPN1 digest to eliminate template DNA from my fragments to prevent the expression of a replicase that would mix selection results by allowing CMR to be expressed.
Draven Rane well, we assumed here that everything is right with the cloning design, with my rich experience, I have cloned 10k plus custom constructs, usually it is design, the reason why you can't clone something. Also these gibson kits contain three enzymes and nobody knows how they work or which one of them doesn't . I never use any kits, so I know exactly what went wrong in why.
- Badges
- Science topic