PCR Amplification of 7kb Plasmid Extraction

 Introduction

In the last GelRed agarose gel containing PCR of genomic bead-beading and 7kb plasmid extraction, smears were detected with the PCR reactions whereas a band at 7kb was seen with one of the other plasmid extractions. This specific sample will be used in a long amplification PCR reaction to prepare for Gibson assembly if a clear band it detected at 7kb on an agarose gel. Furthermore, the GelRed proved to be a sensitive and introspective tool to replace UView dye. 

After PCR, 7kb samples and CmR samples from earlier may need to be cleaned up using the Monarch PCR & DNA Cleanup Kit (5ug) if any genomic DNA is present. This is because Gibson assembly requires a clean vector and insert for calculating pM ratios. 


Methods

To run PCR reactions, two 25ul reactions were prepared with 20ul of Taq1 master mix containing 3M MgCl. MgCl is added to promote specific binding of polymerase to the template. 2 ul of forward and reverse primers for the 7kb plasmid were then added. Lastly, 3ul of the plasmid extraction containing the 7kb plasmid was added to make a total volume of 25ul. After homogenization, reactions were run with the same PCR settings previously. This was then ran on a GelRed agarose gel. 

To clean up all 4 PCR reactions, samples were diluted with a 2:1 ratio of binding buffer to sample. Next, a column was inserted into a collection tube where the sample was then loaded onto. After centrifugation for 1 minute, flow-through was discarded and 200ul of DNA Wash Buffer was added and centrifuged for 1 minute. This step was then repeated, and the column was transferred into a new 1.5mL centrifuge tube. Lastly, 15ul of DNA Elution Buffer was added to the matrix and incubated for 5 minutes at room temperature before centrifugation. these were then run on another agarose gel. 


Results


7kb PCR reactions pre-cleanup


Discussion

After viewing this gel, samples were put through PCR cleanup and run on another GelRed agarose gel. The second gel wasn't stained correctly, and no more samples could be used without risking the volume needed for Gibson assembly. However, in the figure above we can see multiple distinct bands. The top band seems to be at 7kb , with another band above it which could be dimerized 7kb. Below we see bands at ~1750bp and ~2500bp which could be non-specific signaling of the primers. Despite these bands, we're going to continue with Gibson assembly assuming our sample is 1/3 7kb vector. Afterward, we will transform competent cells of E.coli with our Gibson assembly product. 


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