7kb plasmid

 Abstract

The 7000 base pair plasmid is seen within Deinocioccus Aquaticus, this is the plasmid that we wish to use to transform the plasmid and reintroduce it into the same species and then into different species. This can be achieved through different means. 

Introduction

The binding buffer was not a tested variable this week but will be kept in the thought process. The restriction enzyme was used in the exact protocol with a possible less or more than 1ug of DNA. The Restriction Enzyme (RE) is going to be the focus of the process going forward.  

Method

We are using the standard plasmid isolation protocol for this project. Afterward, we are then running the plasmid that we believe we have and doing RE with xba1. This is incubated in the heat block for 2-3 hours. (on this run) 

RE Protocal   For Xbal

RE- 1ul

DNA- 1ug

Buffer- 5ul

Total RXn- 50ul 

Time=?

Thermoscience Kit was reused, without the standard plasmid pre-steps. 

Results

Figure 1: 1% gel with a original protocol of xbal 

Figure 2: Table of nanodrop from plasmid extraction 

Figure 3: plasmid run with primers in the PCR machine

Discussion

There was some inconsistency between the times for restriction Enzyme, when we did the times of; 0hrs and 1 hrs,  showed on a gel though it still showed in the dimerized forum.  We do not know why there is no separation when we have a plasmid extraction. The first time the RE was done there was a slight inconsistency at 2 hours and 3 hrs. So these times needed to be redone. With this RE work, there is also a possible primer to be used. this will work in tandem with the enzyme. If there is nothing at the time of 2 hrs then there will be a possible change in the enzyme for restriction. Since it was shown through figure 3 that we are in fact getting the 7kb plasmid there was the question of why it was not showing on any previous gels. With this new batch of plasmid extractions the thermoscience kit was used, This was shown to work in figure 2, which shows that there was significant plasmid extraction without any kind of lysozyme. Though the fact that there is nothing showing on gels that are from the previous runs is still not explained.