Using New GelRed on Edited 7kb PCR Reactions and Extractions

 Introduction 

Recently, a heat gradient from 50°C-66°C on the 7kb plasmid from D.aquaticus was ran and contained no visible banding. Due to an empty agarose gel, this may reveal that the template we're using ( boiled cells ) doesn't contain the 7kb plasmid. This would mean our primers aren't able to bind appropriately. Another possibility is that our annealing temperature. Therefore, the alternative bead-beating template will be used in a PCR reaction and run on the more sensitive GelRed agarose gel. 

Additionally, a new lysing protocol involving proteinase k and lysozyme will be used to try to extract 7kb plasmid. A theory to test is that we've obtained 7kb plasmid and hadn't seen bands due to the previous dye needing >500ng of template DNA. This new GelRed should only need 5-50ng of DNA to bind visibly. 




Methods

To run LongAmp PCR products, PCR settings were set to the following: initial denaturing was set to 4 minutes at 94°C, denaturing for 30 seconds at 94°C, annealing for 45 seconds at 59°C, extension for 6 minutes at 65°C, final extension for 10 minutes at 65°C, and an infinite hold at 10°C. Two 25ul  reactions were prepared with 20ul Taq 1X mastermix with 3M MgCl added, 2ul of forward and reverse primers, and 3ul of bead-beading lysis template of D. aquaticus from 2-20-24. 

To lyse cells, 3mL of overnight culture of D.aquaticus was centrifuged in two 1.5mL centrifuge tubes. Afterward, the supernatant was discarded. Following this, 300ul of 95% ethanol was added by resuspension. After incubation for 10 minutes at room temperature, the mixture was centrifuged and supernatant was discarded. To further lyse cells, 135ul of 50mM Tris-HCl was resuspended with the pellet. 1ul of T4 lysozyme was added to the mixture. Immediately following this , the tubes were incubated for 10 minutes at 4°C. 7.5ul of 10% SDS and 1ul of Proteinase K were then added and followed by a 10 minute incubation at room temperature. Lastly, this was followed by the Zyppy Plasmid Miniprep Kit, and eluted in 25ul of elution buffer. 

To make the GelRed agarose gel, 30mL of TAE and 0.30g of agarose were mixed in a 125mL flask and microwaved until dissolved. Once cool, the ratio of 10,000X GelRed Dye and TAE was 1ul dye per 10mL TAE. Therefore, 3ul of 10,000X GelRed was added to the mixture and mixed until homogenous in color. The gel was then poured and samples were made with the same ratio of loading dye according to UView dye. 


Results






Discussion

Lanes 3-4 tend to have more density around 7000bp according to the 1kb ThermoScientific ladder. However, there are no visible bands post-PCR at 7kb. The lysing sample in lane 6 shows a distinct band at 7kb , whereas lane 7 is also a smear. We're unsure as to why one plasmid extraction contains the 7kb plasmid, however, this means we can use the sample in the 6th lane as the template for future PCR reactions. Additionally, we should be able to prep for Gibson Assembly following this. 



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