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okay so I'll start out with presenting

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my goal is to find a small efficient

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ligating right design and I hope I know

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you guys know what a ribozyme is it's an

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RNA that has catalytic function and why

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ligation well um you're going to talk

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about origin of life studies you want to

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try to generate more complex products so

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your product should be more complex than

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your starting material and that's what

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ligation is ligation is taking two

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pieces of RNA you're putting them

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together you're like a ting them into

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one product a more complex product so I

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want to increase ligation in a ribozyme

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and I want to try using different

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conditions particularly by freezing and

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then I want to increase that yield by

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introducing thermal cycling or

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freeze-thaw cycles so I won't go very

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much in the background for the RNA world

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I I'm gonna see I'm assuming that

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everyone know this by now um that this

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idea started around the late 1960s and

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it suggested that RNA is the first

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biomolecule as opposed to DNA or protein

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and it's because RNA can store

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information and it also has an enzymatic

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function but the mage one of the major

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problems for RNA is that it's fragile

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and if you ask anybody who worked with

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RNA they'll tell you it's prone to

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degradation so if you heat it it'll

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probably degrade and pray if you have

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something like magnesium in your sample

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um so one solution to that is lowering

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your incubation temperature and when you

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I guess when a lot of people think about

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freezing or ice they think about all the

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water molecules and all the solute sin

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solid phase well that's true if you have

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if you go below this eutectic

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temperature on this phase diagram here

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what we're interested in is this

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triangle area here where the majority of

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the water molecule isn't is in an ice

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crystal but the remaining liquid water

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is mixed with your solute and that makes

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up your eutectic composition and this

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eutectic composition exists in channels

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very small micro meter in width grooves

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and it's shown right here so very small

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tunnels a nice and this provides a

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protective

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for your RNA it concentrates your solute

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and for your RNA you could also reach

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alternative stable conformation that

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wouldn't be possible if you tried

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incubating at higher temperatures and

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and above all your RNA can still retain

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some enzymatic function this is

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demonstrated by by the r18 polymerase

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this this was this is from a paper in

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2010 the r18 polymerase is an artificial

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ribozyme it's lab-created and it's

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outlined in black right here and it

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binds to this template in green and it

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adds nucleotides one by one to this

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primer strand right here so when people

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think about polymerase they think about

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a polymerase that's made out of protein

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and a common one is this t7 RNA

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polymerase right here from bacteriophage

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and it does wonderfully at 37 degrees so

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the higher you see these bands up here

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the higher you go the more the longer

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the projects are but at negative 7 if

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you freeze it you don't see any

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polymerization so how does the r18 fair

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at 37 it's not shown here at 37 it

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doesn't do so well because the

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polymerase degrades but if you start at

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a slightly lower temperature than a room

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temperature it does compare ibly well as

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the protein polymerase but the major

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difference is at negative 7 so here the

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r18 can still catalyze polymerization at

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negative 7 below freezing where the

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protein equivalent you see no product at

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all so this is something that RNA can do

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that the protein can't but this plum

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race is a little too long I don't it's a

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little too big for what I want to what I

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want to work with so this this is what

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I'm actually working with this is the

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hammerhead ribozyme and this was a first

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found in the mid-1980s in plant varese

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this specific sequence though is from

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justice omen which is a parasitic worm

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so this fri design is traditionally

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known to cleave and not like it even

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though they're both reverse

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reactions so in a cleavage reaction you

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would have the enzyme which I outlined

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in red here bind to the substrate in

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blue and it would cleave it or cut into

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two pieces generating two fragments of

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this p1 fragment down here and this P to

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fragment up there in a ligation reaction

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the opposite would happen where the

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ribozyme binds to p1 fragment and the p2

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fragment and it would like it it back

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together back to one strand efficient

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ligation with this sequence wasn't

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demonstrated until two thousand seven

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here the p1 fragment is labeled and if

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it's like ated to p2 then you should see

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this band shift up here and this was

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done at room temperature but it was only

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possible if you added millimolar amounts

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of magnesium and this is a fast reaction

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so here 18 seconds you're done and their

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maximum yield 23% I thought this was a

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great ribozyme to start out with I

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started out with magnesium repeating

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their experiments but I also tried

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calcium and iron and in short they

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worked but that's not the focus of this

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talk although you're welcome to ask me

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about it iron people so what I'm focused

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on is ligation through freezing and if

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you do it this way you don't have to

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have divalent cations in your sample um

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this this is just a shorter example of

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the different temperatures I used and I

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think that C is supposed to go there but

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temperature alone isn't enough so as

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demonstrated here these two lanes right

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here they're incubated at the same

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temperature negative 10 but one was

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frozen before incubation and one remain

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liquid the entire time so even though

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it's the same rank you Batian

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temperature you still have very

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different results this demonstrates that

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you need freezing or the concentrating

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effect to get ligation so next we looked

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at how fast this reaction is and while

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it's not that 18 seconds from that you

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get with magnesium we still get a higher

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final yield and based on this the

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majority ligations done in an hour so we

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decided to use one hour in our thermal

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cycling experiments so thermal cycling

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is freestyle cycling and it's supposed

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to mimic day-night cycles so each of our

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cycles is there's a two-minute heat step

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and then there's an hour long incubation

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free step so that's one cycle here and

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then two cycles you have to heat steps

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and then two-hour-long free steps and

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that wasn't supposed to happen you

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should see like fans up here so our base

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yield what we started out with is

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twenty-five percent but with each

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additional cycle so basically each

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additional day we should get more and

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more like ated product so we thought

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this was a you know great start but

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before going further into cycling

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experiments we were to try increasing

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that base yield that twenty five percent

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so it already briefly went over

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different temperatures you can get

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between twenty-five to thirty percent

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depending on if you're between negative

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20 or negative 10 we tried looking at

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sugars of millimolar amounts of glucose

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sucrose lactose your halos well it

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didn't help it didn't hurt we tried

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looking at different buffers pipis

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sodium phosphate Tris and as long as

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they were the same pH they were

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comparable pH pH is important so our

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standard is ph 8 and if you go a little

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higher it didn't change the a yield but

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if you go lower it'll start decreasing

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that yield and if you go about 5.5 it

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just killed the reaction there's just no

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ligation going on now amino acids now

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why would I be talking about amino acids

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if if this is in the context of the RNA

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world well let's face it I doubt early

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Earth was as clean as our test tubes so

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we decided to look at what would happen

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if we added or contaminated our samples

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with amino acids so we tried looking at

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the 20 standard amino acids and I didn't

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list all of them but 18 out of 20 fell

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in this category here none of them

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increased the ligation yield but none of

202
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them dropped it below twenty percent

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only two of them drastically reduced our

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yield which is tryptophan and isoleucine

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and overall I think this is good news

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because the the earliest amino acids at

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least the ones that are proposed

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earliest amino acid they're on this side

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of the list and the last thing we tried

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was anions this might sound a little

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strange because with RNA studies usually

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focus more on cation cation effects and

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that's because RNA is negatively charged

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cations are positive so you expect

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interactions we didn't think that anions

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would contribute very much but well we

217
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were wrong so depending on which sodium

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00:09:46,360 --> 00:09:43,370
salt you used you get very different

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results our best ones were from acetate

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for weed and citrate and we think it has

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something to do with their carboxyl

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groups fluoride chloride bromide and

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iodide they seem to follow some general

224
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patterns like fluorine and chlorine are

225
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supposed to be like more electronegative

226
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than bromine and iodine they're also

227
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smaller in size then bromine and iodine

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and in the context of chemical abundance

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there's a lot more fluorine and chlorine

230
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than bromine and iodine and we also took

231
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this as another piece of good news

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because based on this we don't want a

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lot of bromine and iodine around we're

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still not sure exactly how the anions

235
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are affecting the the ligation yield but

236
00:10:31,810 --> 00:10:29,480
we but we are exploring some ideas so

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that's where we are experimentally I'd

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like to end this talk by summarizing

239
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that ice preserves RNA and it also

240
00:10:43,150 --> 00:10:40,190
preserved an enzymatic functions of your

241
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RNA particularly ligation what we're

242
00:10:49,150 --> 00:10:45,890
looking at and you don't need to die

243
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valent cations to do it and it this

244
00:10:54,579 --> 00:10:51,250
ligation reaction also tolerates the

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majority of amino acids the osmolytes

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that we looked at and you can try using

247
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different buffers in different salts and

248
00:11:04,500 --> 00:11:01,490
you can still get ligation you can

249
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increase the base ligation yield by

250
00:11:07,630 --> 00:11:07,190
freeze-thaw cycles and really this last

251
00:11:09,760 --> 00:11:07,640
point

252
00:11:12,550 --> 00:11:09,770
our next step it's to combine thermal

253
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cycling with our best ligation yield and

254
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that was that forty percent from acetate

255
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so we want to get above that twenty

256
00:11:33,700 --> 00:11:19,280
percent which is our best so far so

257
00:11:35,890 --> 00:11:33,710
that's it that was really cool have you

258
00:11:38,320 --> 00:11:35,900
given any insight into how this might

259
00:11:40,510 --> 00:11:38,330
vary with pressure because both the

260
00:11:43,270 --> 00:11:40,520
pennsbury I hypothesis and the idea of

261
00:11:46,570 --> 00:11:43,280
delivery of volatile to the earth could

262
00:11:48,640 --> 00:11:46,580
mean that if RNA is kind of having fun

263
00:11:51,610 --> 00:11:48,650
ligating itself on comets for a long

264
00:11:54,970 --> 00:11:51,620
time it could be safe there um I don't

265
00:11:56,860 --> 00:11:54,980
know about low pressure but I did look a

266
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little bit into very high pressure and

267
00:12:04,300 --> 00:12:00,280
that could that could actually affect

268
00:12:07,030 --> 00:12:04,310
the way that ice is formed so this this

269
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type of ice it's normal pressure and

270
00:12:13,390 --> 00:12:11,720
it's what phase 1 H ice but with high

271
00:12:16,360 --> 00:12:13,400
pressure you get different types of

272
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phases of ice I and I don't know I've

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never I haven't tried different

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pressures it's just what was in our lab

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so going off of the pressure in the ice

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bit you were mentioning that this

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reaction only occurs under these frozen

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conditions where you have the solid

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phase and you're attributing that to an

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increase in the concentration so we

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reason to think it might be an effective

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surface chemistry with the ice um we

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don't know if like for instance we don't

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know if the RNA is somehow like

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concentrating near the surface the the

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the walls of the of those tunnels we

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don't know so far all we know is that it

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is there is a concentrating effect and

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you can visibly see that if you stain it

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if you stay in the RNA and you detect

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fluorescence but