Gibson Assembly Attempt with 7kb and cmr

 Introduction 

Previously, PCR amplification was run on 7kb plasmid extracted via the Zyppy Kit and cleaned up through the Monarch PCR & DNA Cleanup Kit (5μg). When viewed on a GelRED agarose gel, 3 bands were present, 2 were non-specific to the 7kb we wanted. However, we will continue to attempt Gibson assembly using 3 times as much vector to make up for 1/3 of our sample being the 7kb plasmid. Afterward, we will transform competent E.coli cells with the Gibson assembly product. 


Methods 

To attempt Gibson assembly, we began by diluting our 46ng/μl cleanup sample of cmr to a concentration of 11.5ng/μl. We diluted our sample to keep our vector and gene equimolar. Next, on ice , we added 9μl of our 7kb samples to 1 PCR tube each. 1μl of our diluted cmr sample was then added to both tubes, making a total volume of 10μl in each. Next, 10μl of Gibson assembly mastermix was added to each tube. Our positive control had 10μl of control reagent and 10μl of Gibson assembly mastermix. All tubes were  placed in the Thermocycler and incubated at 50°C for 15 minutes. Lastly, samples were stored at -20°C.


To attempt transformation, competent E.coli cells were thawed on ice. We then chilled 2μl of ligation mixtures in 2 1.5mL centrifuge tubes. Next, we added 50μl of competent E.coli cells to the DNA and mixed via resuspension and flicking. These mixtures were then placed on ice for 30 minutes. To heat shock, cells were placed in a 42°C water bath for 30 seconds then placed on ice for 2 minutes. Immediately, 950μl of LB media was added to each tube. We then placed these tubes in a shaking incubator at 37°C for 60 minutes. Lastly, 50-100μl of our mixtures were spread on pre-warmed selective plates. Our 4 variant selective plates had LB with no antibiotic , chloramphenicol at 25μg/mL , chloramphenicol at 170μg/mL, and ampicillin at 50μg/mL. Our 4 variants were our positive control mixture, our negative control containing no plasmid, and our two experiments containing different extraction samples. There were a total of 15 plates streaked with 50μl



Results 

Gibson Assembly Plasmid #1 , no growth on chloramphenicol plates



Gibson Assembly Plasmid #2, no growth on chloramphenicol plates


Postive control, growth on Ampicillin and NA plate


Negative Control, no growth on antibiotic plates, growth on NA plate



Discussion

No growth of our Gibson Assembly transformations were seen on chloramphenicol plates. Therefore , we can conclude something went wrong during the Gibson assembly reactions as E.coli cells are extremely competent. Our next steps would be to find a more suitable PCR reaction. Our 3 bands after PCR cleanup likely are the reason we weren't able to have successful Gibson assembly. Finding better PCR settings would ideally require a heat gradient mixed with variable MgCl additions. However, due to lack of plasmid extraction template, four reactions will be ran. 





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