Various Lysing Methods with D.aquaticus 7kb Primers

 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 amplification PCR reactions, 20ul of long amplification mastermix is added to the 4 pcr tubes. Then, 1ul of forward and 1ul of reverse m23 primers are added to 2 tubes. We then added 1ul of forward and reverse 7kb primers to the other 2 PCR tubes. Next, 100ul of D. aquaticus culture is boiled for 15 minutes at 95°C in a thermal cycler. After setting the culture on ice for 1 minute, 3ul of the boiled culture is added to 1 tube containing 7kb primers and 1 tube containing m23 primers. 3ul of the plasmid extraction template is then added to the other reactions for a total of 4 25ul PCR reactions. Lastly, PCR reactions were read on the nanodrop spectrophotometer and ran on a 1% agarose gel with a 10:2 template to dye ratio. 


Results








Agarose gel containing bands at ~700 bp




Discussion

The m23 bands showing at around 700bp indicate that our master mix isn't a current issue. Additionally, boiled cells may be a preferred template, as the band is significantly brighter than the plasmid extraction band. Despite lowering the annealing temperature, no bands at 7kb were seen for either of the templates used. Two hypotheses are that our initial denaturing time isn't long enough or that the annealing temperature isn't in the correct range. To test these hypotheses, we will extend our initial denaturing time from 30 seconds to 4 minutes, as well as perform a heat gradient PCR reaction with boiled cells as our template. The range that we'll run our heat gradient by is 50-66°C. Bands will indicate whether or not our 7kb primers are constructed correctly.


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