Of the twelve flasks (collectively containing bacteria from 32 colonies) about half looked definitively negative. We are performing plasmid isolations on four of the more positive-looking subcultures.
Will run these along a gel when the isolation is complete and see if there is any DNA. If so, we will probably send the plasmids out to be sequenced.
The results of the most recent sequencing were mostly positive. 4/6 were what we wanted.
As of today, the plasmids we have are:
- mt2S
- mt4S
- mt2S4S
- mt5P
- mt5S
- mt1P
- mtTAT
The plasmids we need are:
- mtP
- mtCAT
- mtTAC
In the big experiment, we are likely to include mtPG as well.
Today we will conduct a repeat transformation using 5uL of the recombinant mix with mtPG as a positive control.
UPDATE: July 4th - A handful of colonies on mtCAT, which we suspect are background. mtPG was overwhelmingly positive, as anticipated, but mtP and mtTAC were depressingly negative.
May subculture mtCAT to Amp broth just in case (My idea!).
Dr. Miller spread the leftover cells onto a couple of Kan plates. Colonies grew for 5P, CAT, and no insert. After a couple of days, colonies also grew for TAT.
We completed the higher oligo concentration transformation with great success:
27 colonies for mt1P
18 colonies for mt5S
Today (the 19th) we are doing plasmid isolations for mt1P, mt5P, mt5S, mtTAT, and mtCAT.
Update: Only the mt5P worked. The other plasmids were Ymt.
After celebrating the success of yesterday's gel, we mixed together the seven remaining primers with the vector.
These are the primers with which we are transforming today:
mtP, mt1P, mt5P, mt5S, mtTAC, mtCAT, mtTAT, no insert (control)
The contents of these tubes are as follows:
- 5Ī¼L primer mix
- 5Ī¼L vector
- 10Ī¼L 2X master mix
These were incubated at 50Ā°C for 30 minutes, and then 5 Ī¼L were extracted from each for use in transformation.
Not much can be concluded until we see the results of todayās transformation, but I am VERY excited to see what the plates have in store for us tomorrow.
A Couple of Thin Red Lines on a Gel Can Really Make Your Day
Dr. Miller removed the samples at 6 PM (incubation time: 1 hr, 40 minutes) and ethanol precipitated them. There is a good chance the ethanol will have denatured the enzymes (EcoRV and AgeI). Also, even if the enzyme persisted, most of the magnesium will have been removed during the process. As such, we will not be heat-pulsing to inactivate the enzyme.
We spun the samples for ten minutes at 15000 rpm, but did not observe a pellet, which was worrying at first. We dried them and resuspended in 50ul of 1X TE Buffer. These were run side-by-side on a gel with a standard ladder.
I then prepared (labelled) tubes for the next set of transformations. Here is the current status for ALL of my primer mixes, as well as the results of the gel:
Conclusion: Digest worked perfectly! Single bands for both of the samples, and no smearing. Smearing would have indicated a problem with the buffer, as we had before, and the fact that we have single bands means that all the DNA has been linearized. If the digest had not occurred, we would probably see two bands - one circular, one supercoiled. Time to celebrate!
A Couple of Thin Red Lines on a Gel Can Really Make Your Day
Dr. Miller removed the samples at 6 PM (incubation time: 1 hr, 40 minutes) and ethanol precipitated them. There is a good chance the ethanol will have denatured the enzymes (EcoRV and AgeI). Also, even if the enzyme persisted, most of the magnesium will have been removed during the process. As such, we will not be heat-pulsing to inactivate the enzyme.
We spun the samples for ten minutes at 15000 rpm, but did not observe a pellet, which was worrying at first. We dried them and resuspended in 50Ī¼l of 1X TE Buffer. These were run side-by-side on a gel with a standard ladder.
I then prepared (labelled) tubes for the next set of transformations. Here is the current status for ALL of my primer mixes, as well as the results of the gel:
- mtP (No transformants, redo pending...)
- mt2S (Success! We have plasmid.)
- mt4S (Success! We have plasmid.)
- mt2S,4S (Success! We have plasmid.)
- mt1P (Transformation pending...)
- mt5S (Transformation pending...)
- mt5P (Transformation pending...)
- mtTAC (Transformation pending...)
- mtCAT (Transformation pending...)
- mtTAT (Transformation pending...)
Gel:
(Left to right: Ladder, Cut Ymt in Yellow Buffer (NEB 1.1), Cut Ymt in Green Buffer (CutSmart).)
Since we ran out of U1 with which to construct our new plasmids, weāve decided to cut up some Ymt. However, the digest we did was quite strange - on the gel, it looked terribly, terribly smudged, including the sample that contained buffer but no enzyme!!!
Today, we are repeating the digest with green (CutSmart) and yellow (NEB 1.1) buffer, but we think itās not the same CutSmart as last time.
The tube composition is as follows:
Tube 1:
45Ī¼L of Ymt plasmid
5Ī¼L of CutSmart
1Ī¼L of AgeI
1Ī¼l of EcoRV
Tube 2:
45Ī¼L of Ymt plasmid
5Ī¼L of 1.1 buffer
1Ī¼L of AgeI
1Ī¼L of EcoRV
These tubes went into the 37Ā°C bath at 4:20 PM. I must leave the lab early today, as I am moving out of my apartment, but Dr. Miller is to be returning from his seminar on malaria around 5:45 PM. As such, the digest will run for approximately 1 hour and 25 minutes.Ā
Personal aside: I am concerned about the green buffer, and hope desperately that it works in this particular digest. The yellow buffer is decidedly lacking in the efficiency department, and Iād be significantly bummed if we had to use it for the remainder of the experiment. Then again, Iām told CutSmart isnāt terribly expensive, so, who knows. Maybe if it doesnāt work, we can just order some more!
Iāve been feeling like dead weight this entire time, unable to help. All of that changed within just a few hours.
I was leaving my human genetics class (which I didnāt enjoy because the professor irritated me to no end) and figured, hey! Iāll see whatās up in the lab. It was just me, one other student, and Dr. Miller. Well... They let me mix the lysogeny broth! Not only that, but I got to pour it! I know itās minor stuff, but... I feel like Iām making a real contribution for a change. That means the world to me... Pics from today under the cut!
In hindsight, I should have started documenting these things a long time ago. Iāll start as early as I can.
I became interested in genetics at a very young age. I always knew it was the field for me, and Iām not ashamed to say my passion came from a humble beginning. It began with a videogame.
Creatures is the best artificial life simulation Iāve seen to date. And I use the phraseĀ āartificial life simulationā out of habit, because if I call it a game, people take it far less seriously. It is, in essence, a PC game, but it is also so much more. I wonāt go into the details - you can check out the game for yourself - but it was my gateway into the realm of genetic research and experimentation. In a simulation, where you arenāt limited by bioethics, you have full control over your experiments, and I went wild.
It was several years before I started creating my own breeds for the game - not due to lack of experience with the genetics kit, but due to lack of patience when spritemaking. To date, Iāve only ever made breeds that stay small their entire lives because I canāt be bothered to make larger sprites.
So, thatās how I began my journey into genetic research. A videogame. Pretty amazing how far Iāve come...
Now, that brings me to highschool. I was in the infamous IB program, and very nearly didnāt get the diploma due to a huge mix-up in French. It was very stressful, and the rejection from MIT didnāt help at all.
MIT was always my dream school. But, I knew the odds werenāt great. I also applied to Stanford, Harvard, and Yale. I figured, hey! Iām plenty good enough. At least one of them is bound to accept me. All of them turned me down. That stung. I did get accepted to UTD, my backup school. It didnāt seem like much (although theyāre expanding very quickly Iāve found, so they might be a bigger deal now depending on when youāre reading this).Ā
So now Iām here. At UTD. At least, thatās where I am at the time Iām writing this. Things got off to a slow start. I was accepted into a research team with Dr. Dennis Miller, but I felt like such dead weight for the longest time. All of that changed today (November 16th, 2017).
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