S-STEM Scholar Aland Ali's Blog

 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...

 Introduction  Recently, we had obtained E. coli's cm^r gene from pRAD1. However, when observing a gel for 7kb fragments, only primer bands were shown. Three hypotheses were that the master mix wasn't made correctly, the annealing temperature during PCR was too high for binding, and the primers could have been constructed incorrectly. To test these hypotheses, we'll run four long amplification PCR reactions. The two templates we'll use are boiled D.aquaticus cells and a plasmid extraction sample of D.aquaticus that was used when first constructing the current 7kb primers. Each pair will use m23 primers and 7kb primers. The purpose of the m23 primers is to be used as a control for master mix. The m23 primers are expected to prime to the m23 portion of D. aquaticus' 7kb plasmid and show on the agarose gel. We'll also lower our annealing temperature to 57 °C and expect more non-specific binding than from our 59 °C samples.  Procedure To begin making our 4 25ul long...