Continue on overlap and restrictions of linear plasmid

 This week Monday started off with Friday's work and running our  Anna, Hilgarth, and lactonase sadly when we ran that through a gel we didn't get anything out of it. It was very unexpected to not see anything since our lactonase was our positive control and was 99% going to show up. 

The first one is a ladder, space, Lactonase, anna 1, anna2, space, hilgarth 1, and lastly hilgarth 2

We decided to move on and amplify the left, tet, and right want to see if we could get a better vision of the final fusion. We had one test tube with undiluted primer and one with diluted primer in a gel since we want to see 2700 base pears. since we waited for that to run I worked on getting more experience and making more 1% gels for later lab sessions to be quick and ready then we stored those. The result of this amplifying failed again we had a band stuck in the well and a streak the reason that it could possibly be stuck is that we have a big piece of DNA that wasn't able to come out. We figured that if we loaded more proportions then we should be able to get a clear view of any bands. which is 20 ul left and tet and then 30ul of right to get a more precise view of how clean they are. We cleaned all the gels to get all the junk out which didn't quite do anything but lost a lot of DNA and only had about 3ng/ ul of left and tet. 5ng/ul of right. so we made a reaction tube of 50ng of each fragment and put it through overlap reactions at 37celcius. We want to take around 4 ul from the reaction above and amplify it with an A and F primer. I worked on making more gel to practice pipetting in wells since I've only observed people and it was very fun I realized I really do like to work with the pipettes. So I worked on that and loading the gel with distilled water and dye which we put 60ul of distilled water and 12ul of dye. I also worked on refilling all TAE Buffer and just worked on refilling pipette tubes and cleaning the lab down a little. 

Comments

  1. EricaOctober 17, 2022 at 5:46 PM

    Very nice blog, but don't forget to cite your sources.

    ReplyDelete

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