 Hey everybody, I just wanted to walk you through some of the basics of the OER LibGuide project Which is essentially a a gene splicing project and so you can see on my table here that I've I've done a couple steps already. I printed out the the DNA sequence Which is separate? The plasmid sequence which if ideally you would do it in a different color paper, which I've done here and Then the restriction enzyme page and then you're going to go ahead and cut all those out and So you can see I've done that here, and I keep my strip I keep the name at the top and I cut it off flesh at the bottom And I do that for both the sequence and the plasmid and Then you do it for your restriction enzymes You can just cut around those as well. Make sure you keep the names on top because you'll need those And now you'll have to put everything together Before you can do the second part and so to do that come back over here way You have to make sure you keep your strips in order and so I have strip one and strip two and Strip two will just go on the bottom of one Like so and then you tape them together and you will continue on and do that For the whole strip and this will make one long strip and then when you do your plasmids You will eventually wrap strip three back around to strip one. So we've got three strips here We'll do one two Three and then it'll wrap around to make a big circle. And so I'm going to take a moment Off camera here and do those and then I will walk you through the next step Okay, and now everything's taped together and your enzymes are going to stay apart You are going to have the plasmid in a loop That's how it is in nature in bacteria. It's a small loop of DNA And then you're going to have one long strip. You might want to do this Like I did on a table or on the floor. So now you are ready to determine which enzyme of These eight that you are going to use to make the necessary cuts And so you are going to have to compare them all to both. We'll do the first one And so you come down to the beginning this is strip one and You just set your enzyme next to it and you just move along one at a time Until you find a sequence that matches up exactly to what you got here on the left and right So the left and right here will match the left and right on here And I've actually already done this one. So again, you just kind of go down Until you find a sequence That matches which we have Right there And so then you draw a line with a pencil just as I've done that matches the line that's drawn on the enzyme And then you label it And then you will continue on For the duration of this and I've only done one but you could see this One time only you could see it more than one time two three times the goal ultimately is to find the enzyme or enzymes that cut this strip at least twice and I come back here you want to make sure if I go along here You'll see right there That starts to be bold type that bold type represents the insulin gene that we want to cut out So we want an enzyme that cuts it up here somewhere Does not cut anywhere in the bold if you're doing an enzyme and it cuts in the bold type You can put it off to the side because it's automatically eliminated at that point. It's the wrong one and so then again, we go on to the end and And Bull type ends right there and so our next cut cut Line needs to be after the bull type So we'll do all eight enzymes I'm not gonna do them all but you'll do all the enzymes and you'll have your marks And then you want to find the one or ones it's gonna be at least two I will go ahead and tell you that cut this Once before the gene the bold type And then once after the gene any enzyme that doesn't do that that only cuts once that cuts through the bold type Can be eliminated then you'll take those same restriction enzymes and Move them and compare them to the loop the loop is a little harder But again, you just kind of run them along start at the top of one and Find your matches and draw those lines And ultimately let me spin this again. There are bull type areas here See them there. That's a replication origin. That's gonna be important. There's a question on that That's antibiotic resistance and there's an antibiotic resistance There's well if any of the enzymes cut any of the bold on The sequence or the plasmid you can eliminate it and we want an enzyme that'll cut this one twice And this one once it'll be the same enzyme in both cases. So if it is XMA, and I'm not gonna say it is or isn't XMA would make two lines on this one again above and below Gene and one cut here anywhere except through the bull Letters because we need those intact for replication and isolation All right, and so when you find that you will actually make cuts On the paper, which I'm gonna do next so I will be right back Okay, so after identifying my enzyme and made my lines I went ahead and made cuts and you notice their staggered cuts this point off here Is known as a sticky end and that is used to splice our gene into the plasmid The goal is to make again now one Big loop and so what you will do Is not drop your zone Is you will Tape these together now in yours if you know your base pairing rules, you notice mine don't match up I just made something up to show you kind of how the ends would look but yours The bases should line up and you will tape that into place and then you'll swing around and do it on the other end So go ahead and do that real quick, and then I'll come right back and show you the end Okay, so I have taped everything together. And so now what we have is one big loop And in essence you are done at that point with the gene splicing portion. There are Three pictures you need to take to send to me You'll need to take a picture of your first splice You need a picture of then your second splice and a picture of The actual enzyme that you use so one picture of one splice Second picture of the splice and a picture the enzyme ideally you would insert these and shrink them into a single page work document I know that doesn't always work. So you can if necessary submit the three pictures directly Okay, there is another part of this. I want to show you it doesn't involve the gene splicing directly But let me pause the video again, and I'll be right back to show you how to fill out the table Okay, the table question number seven table number seven You don't need the actual loop for it. You can use it I usually think it's easiest to just reprint the DNA sequence. You do not need the plasmids It's the same DNA sequence that we cut and tied together, but this point we're only concerned with The insulin gene and so I've actually went ahead and used just a pencil to make a big mark to know where it starts And then I came over here and mark when it stops In addition, I didn't do them all but I started making marks every three base pairs So one two three mark one two three mark one two three When transcription takes place it works in groups of three to build The messenger RNA codon or the mRNA codon that you're going to use on your table So if we take the first one here, we have ATG and TAC and Those on the table. I've given you I've already filled it in you can see ATG and TAC If you then use your base pairing rules to go from DNA to RNA The Team is with a a is with you and see with G you get a UG Which I've done here and I went ahead and did this like one for you GCC And I had you do one extra step yourself. So the first one I gave you the amino acid I'll tell you how to get that in a second on the next one I stopped short so you'll look this up and then you can see Each time I gave you less and less until I gave you nothing. So that'll get you through the first four Get you started and then you will pick up on your own. So number five on your list would then be ATG again Which we had first and it can repeat amino acids repeat all the time And you would fill in your first two columns with what you have here And then use your base pairing rules to fill in the messenger RNA codon So it may be easiest to fill out all of column one and two and Then you go from two to three using your base pairing rules And then you will go from three to four using the codon table I mean no acid table that's in your textbook. I've provided the number for you in the instructions But if we go back here real quick, we can see our first mRNA codon is AUG So we want those three letters and so you can see here It says first letter second letter and third letter So the first letter was a so that brings us to this row here The second one was you and Then you can see the G so AUG is the codon that represents the amino acid. That's abbreviated MET. It's methionine You don't need to know that you just put MET in the table So if you're watching this we'll go ahead and do the next one which is GCC So go over here our first letter is G Go to the top here second column is C and then we find GCC and it's amino acid is ALA, which is alanine So over here in your table you would put ALA and then you would continue on so for GAC You would have C U G and you would come over here and look up C U G and that's LEU so you put LEU right there That's leucine and you would continue that throughout the entire table Both sides all I believe there are 59 Amino acids I didn't print the whole table for this don't forget to put your name on it And if you have any questions or want me to check any of your work before you submit it Go ahead and just send those to me and I will do my my very best to answer them Do not forget questions one through six. Those are answered through the LibGuides, which the links are provided for you under the project You can try and look them up online, but the the easiest answers They're they're written from the LibGuide project or the LibGuides And so that's where we would like you to go to get those answers Because then you're guaranteed to get the answer we are looking for so again if you have any questions Just let me know thanks