Restriction Enzyme on Deinococcus Aquaticus 7kb Fragment

 Introduction

Last week in lab, two largely concentrated samples of the 7kb fragment from Deinococcus Aquaticus was obtained. However, the gel showed this sample to be at 14kb. A theory for this is that two 7kb fragments are mineralized together. Therefore, we are using restriction enzyme to demineralize these fragments and have them appear at 7kb. If this isn't the case, we will use primers. Restriction enzymes are sequences that are recognized by restriction enzyme to cut plasmids. We cut at the restriction site xbaI (1188 base pairs) but this should show as 7kb on the gel. 

Procedure

A 1mL tube was filled with 68ul pcr water, 10ul cutsmart buffer, 2ul restriction enzyme , and 20ul of our plasmid isolation samples from the week prior (2mg). Once we have a 100ul total , we will incubate this tube on a heat block at 37 degrees celcius. Due to not knowing how long this enzyme will take to completely cut at all of the restriction sites, we will use 4 incubation time periods. These time periods will be 0-3 hours with an hour for each tube after the 1st (0 hours). 4 microcentrifuge tubes were labeled 1-4 and tube 1 was immediately filled with 25ul of our solution and placed on ice. After the solution had incubated for an hour, 25ul was pipetted out and into tube 2, which was then placed on ice. Once another hour of incubation had passed, 25ul was taken out and put into tube 3, which was also stored on ice. Finally, after another hour of incubation, the last 25ul of solution was pipetted into tube 4. Samples 1 and 2 were  ran on a 1% agarose gel a day prior to samples 3 and 4 due to incubation time periods. 

Results



Discussion

The bands we were hoping to see (7kb) didn't show up for tubes 3 and 4. We aren't sure why this is , as more time to cut at the xbaI site should've allowed for only 1 band to show up at 7kb. This is especially confusing because tubes 1 and 2 had bands at both 14kb and 7kb with less incubation time. We think the best way to move forward to confirming this plasmid would be to use primers and pcr after performing more plasmid isolations. 

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