Continuation of Deinococcus Aquaticus 7kb Plasmid Isolation

 Introduction

Last week in lab consisted of many plasmid isolations on Deinococcus Aquaticus. We've switched to using the ZymoPURE Plasmid Miniprep kit and have occurred many problems with concentrations since. Typically, readings of our plasmid ranged around 30ng/ul, but using this kit we've had concentration readings as low as 1ng/ul eluted in 25ul of elution buffer. We've also experienced problems during the neutralization stage of this kit. Normally, our solution should turn yellow with yellow precipitate almost instantaneously after inversion. However, our solution has stayed a pink color and formed some yellow precipitate. We predict that something in the resuspension stage is causing this issue. More specifically, we determined that our resuspension fluid is too diluted in our lysozyme, leading to cells not opening up properly. We are going to test this by having two samples straight from pellet to kit, and two other samples incubated in a lysozyme resuspension fluid mixture, then moved to kit.

Procedure

To begin, 3ml worth of pellet from Deinococcus Aquaticus was put into 4 tubes ( Labeled A B Ae Be) each . One tube was only filled with 2ml worth of pellet (B) before we ran out of culture. These then had been washed with 500ul of multibuffer solution and incubated for 5 minutes. This step was repeated after centrifuging and discarding supernatant. Then, tubes Ae and Be were washed with a mixture containing 40ul of a 10ng/ul lysozyme stock and 500ul of our ZymoPURE P1(Red) solution. These were then put on a heat block at 37°C for 30 minutes.

While tubes Ae and Be were incubating , we suspended tubes A and B in 250ul of our ZymoPURE P1(Red) solution. We then added 250ul of ZymoPURE P2(Blue) solution and inverted 8-10 times and let incubate for 3 minutes. Once our solution was clear and purple (indicating our cells were completely lysed). Immediately after, we added 250ul of our ZymoPURE P3(Yellow) solution and inverted our samples until our samples turned completely yellow. Our samples turned yellow instantaneously this time, indicating our cells had been neutralized this time. We centrifuged our tubes for 5 minutes and transferred 750ul of our supernatant to clean tubes. After this, we suspended 325ul of ZymoPURE Binding Buffer thoroughly into our new tubes. We prepared two Zymo-Spin II-PX Columns into collection tubes and transfered our entire mixture from the previous step into each corresponding tube. After letting our tubes sit for a minute, we centrifuged them for a minute and discarded flow-through. We then added 800ul of ZymoPURE Wash 1 into our tubes , centrifuged them for a minute, and discarded flow through. We repeated this step with ZymoPURE Wash 2. We then added 200ul of ZymoPURE Wash 2, centrifuged for a minute, and discarded flow-through once more.  Next, we spun for an additional minute to get rid of any residual wash buffer from the previous step. Lastly, we transferred our columns into clean 1.5ml tubes and added 25ul of ZymoPURE Elution Buffer directly on top of our frit, incubated for two minutes, and centrifuged for a minute to elute plasmid. Tubes Ae and Be received the same protocol starting at the stage of adding 250ul of ZymoPURE P2(Blue) solution.

Results

After eluting, samples were nanodropped for concentration. These were the following results:

ng/ul A260/280 A260/230

A 3.3    15.74         -1.65

B 6.2          2.04          4.33

Ae    17.6  1.94            2.00

Be 87.7          1.79                1.77



Discussion

Tubes A and B were not good samples. This may be mainly due to not being washed or incubated in any lysozyme before moving to the kit. Tube Be had the best concentration we've had so far with this kit. The purity isn't very good, and we were confused as to why there was a major difference between tubes Ae and Be. We think this may be due to user error or chromosomal crossover. To test this, we will run these samples on a 1% Agarose gel. More Deinococcus Aquaticus was also inoculated in TGY for future usage,

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