Plasmid Preparation

EXPERIMENTAL OBJECTIVES

To demonstrate the validity of Transcriptic’s Autoprotocol-implemented Plasmid Preparation Quick Instruction.

Methodology

E. coli (Mix & Go Zymo5ɑ, Zymo) with pBabe-puro-encoded [1] ampicillin resistance were grown and then processed for DNA extraction and purification using the Plasmid Preparation Quick Instruction.  As a comparison control, the same E. coli strain lacking plasmid-encoded resistance was similarly grown and processed.  DNA yields were determined using a NanoDrop Lite Spectrophotometer (Thermo Scientific).

Results

The Plasmid Preparation Quick Instruction was employed to successfully inoculate and propagate bacterial stocks with successful, high-yield (Figure 1) DNA extraction.

Figure 1  Yields of DNA after extraction and purification.  Bacteria were incubated overnight in ampicillin-containing LB at 37°C with agitation to allow for expansion.  Control bacteria (lacking ampicillin resistance) failed to grow and generated background yields of DNA.  For minipreps, a total of 35 µl of DNA solution was prepared; for maxipreps, 500 µl -: negative control bacteria. +: plasmid-containing, ampicillin resistant bacteria. 

Figure 1  Yields of DNA after extraction and purification.  Bacteria were incubated overnight in ampicillin-containing LB at 37°C with agitation to allow for expansion.  Control bacteria (lacking ampicillin resistance) failed to grow and generated background yields of DNA.  For minipreps, a total of 35 µl of DNA solution was prepared; for maxipreps, 500 µl -: negative control bacteria. +: plasmid-containing, ampicillin resistant bacteria. 

Figure 2  Five microliters of plasmid DNA solution was linearized with BamH1 in FastDigest buffer (both Thermo Scientific) at 37°C for 60 minutes.  DNA was separated on a 2% agarose gel and visualized.  Linearized DNA at expected size (~5.7 Kbp) was observed in the plasmid-positive bacterial preparations. -: negative control bacteria+: plasmid-containing, ampicillin resistant bacteria. 

Figure 2  Five microliters of plasmid DNA solution was linearized with BamH1 in FastDigest buffer (both Thermo Scientific) at 37°C for 60 minutes.  DNA was separated on a 2% agarose gel and visualized.  Linearized DNA at expected size (~5.7 Kbp) was observed in the plasmid-positive bacterial preparations. -: negative control bacteria+: plasmid-containing, ampicillin resistant bacteria. 

 

[1]  Morgenstern JP, Land H. Advanced mammalian gene transfer: high titer retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line. Nucleic Acids Res. 1990;12:3587-96


VALIDATION DATE:  September 2015