Confirmation of Gibson Assembly Reaction and Primers

 Introduction

Previously, transformation wasn't successful with Gibson assembly product. This led us to hypothesize that primers may have too much non-specific binding due to not having ideal annealing temperature and MgCl concentrations. To test this, we will run four PCR reactions. A pair will be ran at 59°C and at 61°C. 7kb template used previously will be ran with variable MgCl concentrations. This will confirm ideal MgCl concentrations as well as ideal temperature for annealing during PCR. 


Methods

Our first pair of Longamp PCR samples will contain 1M MgCl and 3M MgCl, Longamp Mastermix, and 2ul of template. Initial denaturing was set to 4 minutes at 94°C. Denaturing was set to 30 seconds at 94°C. Annealing temperature was set to 59°C for 45 seconds. Extension time is 6 minutes and 1 second at 65°C. Final extension was set to 10 minutes at 65°C with an infinite hold at 10°C. 

Our second pair of Longamp PCR samples will contain 1M MgCl and 3M MgCl, Longamp Mastermix, and 2ul of template. Initial denaturing was also set to 4 minutes at 94°C, as well as denaturing at 94°C for 30 seconds. Annealing and extension are combined when annealing temperature is above 60°C, therefore , annealing and extension were combined to a single step at 61°C for 6 minutes and 46 seconds. Final extension was ser to 65°C for 10 minutes, with an infinite hold at 10°C. 

A 1% GelRED agarose gel was prepared and these reactions were ran through electrophoresis. 



Results

Varying PCR Reaction Settings on GelRED


Discussion

As seen above, more specific binding is introduced when annealing temperature is set to 61°C. Varying MgCl levels don't seem to make a difference in resolution or priming of primers. Although priming is more precise at 61°C, it is not accurate. The band we are looking for is at 7kb, whereas the band seen in the 6th and 7th lanes are ~1750bp. Therefore, primers need to be reconstructed or a different type of polymerase needs to be used. Due to the upcoming conference, projects may need to be paused. 



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