Bead-Beading D. aquaticus and Heat Gradient on Boiled D. aquaticus Cells

 Introduction

Recently, a gel was run with various lysing methods performed with D. aquaticus. M23 primers revealed a brighter band with cells boiled for 15 minutes as a template, 7kb primers hadn't shown. A heat gradient will be performed on boiled cells to indicate the best range for 7kb primers to anneal. The range chosen for this heat gradient is 50-66°C , additionally, initial denaturing will be extended from 30 seconds to 4 minutes to allow further separation of cells. 

Additionally, alternate genomic DNA extraction of D. aquaticus will be used as a template for a long amplification PCR at 59°C annealing. M23 (~700bp) and E. coli's crtb primers (~6650bp) will be used as controls for mastermix denaturation and long amplification on larger fragments. The reason for using culture run-through bead-beading is to determine the best template to use is boiled cells, as it was an unexpected outcome. The alternative DNA is D. aquaticus run through the DNeasy Ultraclean Microbial Kit.


Procedure

8 25ul reactions for heat gradient were prepared by boiling 100ul of overnight D. aquaticus culture for 15 minutes at 95°C. 24ul of boiled culture was added to 176ul of master mix containing 7kb primers, then aliquoted into 8 25ul PCR tubes. These tubes were then run through a temperature gradient in the PCR ranging from 50-66°C. Afterward, 10ul from each tube was then loaded in a 1% agarose gel. 

Separately, 22ul of long amplification master mix was added to 3 PCR tubes. The tube with crtb primers had 3ul of pRAD1 extraction from E. coli added. The 2 25ul reaction tubes with 7kb and M23 primers had 3ul added of D . aquaticus culture lysed via the DNeasy Ultraclean Microbial Kit. Both reactions were annealed at 59°C and extended for 6 minutes. These were then run on a 1% agarose gel. 


Results



Samples 1-8 in descending order from 66°C-50°C in lanes 3-10






Samples containing alternative bead-beading template, and crtb primers with pRAD1 extraction



Discussion

Due to nothing showing on our gel containing our temperature gradient, we can assume that primer construction isn't efficient. However, our second figure containing a smear in the 7kb lane indicates otherwise. Additionally, a smear may indicate that our annealing temperature is too low for the primers to work at. Another heat gradient on the alternative bead-beading template may be ideal. Another possibility is that the 7kb fragment is GC-rich, therefore needing more magnesium chloride in our master mix to increase charge and binding. Running shorter PCR amplifications could show that our primers are priming specifically without getting a full product on a gel. Next week, a normal amplification temperature gradient will be run on the bead-beading template with increased magnesium chloride in our master mix. pRAD1's crtb primers at 59°C annealing will be used as a positive control.



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