7kb

 7kb plasmid

Abstract

The use of a 7000 base pair fragment to be transformed and then integrated into another plasmid. Since this is one of the smaller plasmids that are known, the use of this plasmid may be easier for other plasmids to accept and integrate then other plasmids. This project is to find if the 7kb fragment is usable and transformations are able to be done.

Introduction 

It was shown that the broth that wass previously used was contaminated. After inoculating the new broth to use, it was left to grow overnight and then put into a 4*C firdge. Since there was contamination. The added step is that after the overnight culture has grown a gram stain must be performed before moving forward.  

Method

There was a different protocal needed, so it will be Included.  

The following protocal will be used in every plasmid isolation: 3ml of culture is centrifuge into pelletts 1ml at a time. Then adding 500 ul of multibuffer, followed by a 5 minute incubation before centrifuging for 1 min, supernatant was taken off and 500ul of multibuffer with another 5 minute incubation, centrifuge 1 minute,  taking all of the supernatant from that and adding 600ul of lysozyme and heating for 5 minutes in 37*C heat block.

The zymopure plasmid minikit: 250ul of a red solution, 250ul of a blue lysis (invert 8-10 times), 350ul of a nutrilization buffer(invert 5 times).Centrifuge and then take the suppernatant and put through a spin column about 750ul of supernatant. Followed by centrifuge and the 800ul of wash 1, centrifuge,  wash 2 800 ul, centrifuge,  wash 2 200ul, centrifuge,  the take the frit and put in to clean tube, then use the elution buffer and elute in 30-50ul of elution. 

Then a new step that was created the lysozyme will now be at a concentration of 10mg/mL instead of 1, this will be pre added to the red solution of the process. There may be different interactions between the blue solution and the lysozyme. 

Results

Figure 1: gram stain of D. Aquaticus 

Figure 2:gram stain of D. Aquaticus 

Figure 3: table of new protocal nanodrop readings

Figure 4: table of nanodrop readings

Figure 5: table of nanodrop readings

Disscusion

During the process of this new kit and working on the protocal to fit the needs of deinococcus,  there were some changes made. Firstly the new kit was found to have an improper dilution with the 600ul of lysozyme in the sample and so there was a side experiment of seeing if from pellet and straight into the kit would work at all, as Figure 3 showed that was not the case, where we were getting double digit numbers the following concentrations were so low that there was talk of scraping the kit entirely. In Figure 5 it is shown when A-D were from pellet and followed the protocal precisely, which was horrible. Next there was the inclusion of a 10mg/mL lysozyme solution with the resolution of the kit, this was found in Figure 5 to have produced a better concentration then previous results. The step to follow will be to change the amount of 10mg/mL to be addedto the red solution to see if there was a numerical value that would work best.