Tag Archives: setup

KochLab Stuff, Shotgun DNA Mapping, Tethering

Shotgun DNA Mapping Buffers

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I worked with Pranav to make some H2O and D2O buffers for the shotgun DNA mapping experiment. We use a buffer that we call Popping Buffer for our tethering experiment. The name comes from the fact that this buffer was used during Koch’s (and potentially others’) experiments that involved “popping” bound proteins off DNA while unzipping. The buffer is (final concentration):

  • 50mM NaCl
  • 50mM Sodium Phosphate (which is a mixture of dibasic and monobasic sodium phosphate)
  • 10mM EDTA
  • 0.02% Tween-20

We made two 100ml amounts of solution in D2O and H2O respectively. And the buffers were concocted from separately made solutions in each water type:

  • 4M NaCl was made as a solution in both D2O and H2O
  • 500mM Sodium Phosphate (monobasic) in D2O and H2O
  • 500mM Sodium Phosphate (dibasic) in D2O and H2O
  • 100mM EDTA in D2O and H2O

From some Popping Buffer we made BGB (Blotting Grade Blocker) in both D2O and H2O at a concentration of 5mg/ml (about 15ml volume). And we also made aliquots of anti-dig in PBS (from only H2O, since we aliquot 20ul amounts and then add 180ul of Popping buffer when it is experiment time).

D2O adaptation, Microorganisms, Water Type Effects on Organism Growth, Yeast

D2O Adaptation Day 7

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Another day another dollar… or something.

Ahem…

I set up another culture of D2O and 50% D2O for little yeast to grow in: 9ml of D2O YPD mixed with 1ml of culture from generation 2; 3.5ml of D2O YPD with 3.5ml of DDW YPD and 400ul of 50/50 culture from generation 2. I ran out of D2O hence the reason for the smaller volume than normal. Tomorrow I will try a larger culture.

And here are the measurements from yesterday’s growth and today’s starter measurements:

  • Gen 2 D2O – 2.777 at 48h
  • Gen 2 50% D2O – 3.064 at 48h
  • Gen 3 D2O – 0.865 at 0h
  • Gen 3 50% D2O – 0.419 at 0h
D2O adaptation, Microorganisms, Water Type Effects on Organism Growth, Yeast

D2O Adaptation Day 6: Time Trials

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Yesterday’s cultures incubated very well. In just 24 hours the absorbance tripled. I don’t have enough data yet to confirm if that is high, average, or low, but I feel it is pretty decent (which is me taking an optimistic middle ground). Unrecorded yesterday, I started a DDW sample from a single colony for today’s time trial. Let’s check out the stats:

  • DDW – 3.018
  • 50% D2O (this is labeled as 50/50 in the spreadsheet) – 2.776 (started at 0.61)
  • 99% D2O – 1.873 (started at 0.618)

To setup the time trials, I inoculated 1ml of each of the above samples in 9ml of YPD (of each respective type, with 50% D2O being 4.5ml of DDW YPD and 4.5ml of D2O YPD). I will record the growth hourly via the nanodrop.

D2O adaptation, Microorganisms, Water Type Effects on Organism Growth, Yeast

D2O Adaptation: Day 5

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Yesterday I couldn’t come in to check on the progress of my yeast, so today I’m catching up. Tomorrow I’m scheduling a time trial experiment to check the progress of hourly yeast growth. I’m expanding that experiment to include yeast growth in 50% D2O (mixed with DDW).

But that doesn’t mean nothing grew today. So let’s check out the 48 hour growth (going back to Saturday:

  • The original D2O sample – at 120h (5 days) the absorbance is 2.944. Compare that with Saturday (2.497).
  • The 2nd generation D2O sample – at 72h (3 days) the absorbance is 0.062. Compare that with Saturday (0.000)
  • The 1st generation 50% D2O sample was 3.155 at 72h, compared to 0.490 on Saturday.

I also started a new second generation D2O sample. There hasn’t been significant growth in the original 2nd gen sample so I pulled a larger amount of culture from the 1st gen sample and put that in a new batch of D2O YPD:

  • 9ml of fresh D2O YPD mixed with 1ml of D2O YPD culture.

And I followed suit with a second generation of 50% D2O:

  • 5.5ml of D2O YPD, 5.5ml of DDW YPD, and 1ml of DDW YPD culture.

The reason for the extra volume of 50% D2O is that I read the pipette wrong and added 5.5ml instead of 4.5ml of YPD broth. Oh well.

To better track the growth, I took so t=0h readings:

  • 99% D2O YPD (2nd generation) – 0.618
  • 50%D2O YPD (2nd generation) – 0.610

So as you can see, my mistake actually put the starting culture absorbance counts at about the same number. That works well and it’ll help better gauge 24h growth.

Until tomorrow…

50/50-72

D2O adaptation, Microorganisms, Water Type Effects on Organism Growth, Yeast

D2O Adaptation Day 2

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The 48h sample reading is 0.997- recall the 24h sample reading of yeast in DI water was 3.05, three times higher absorption!

I replaced that sample in the incubator to measure again tomorrow. I also inoculated a new D2O sample from that one and will record the 24h and 48h growth of cells in that batch. Finally I inoculated some cells (from the 48h D2O sample) and placed them in a 50/50 mix of D2O and DDW. I’ll record those values tomorrow as well.

D2O adaptation, Water Type Effects on Organism Growth, Yeast

Yeast Adaption Time Trials: Setup

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Yesterday’s starter cultures finally started to grow so I can move ahead as planned. For now there isn’t much difference between these experiments and the time trials I did in May (see D2O1 and DDW1 categories). The only difference is that I’ll be comparing the growth amongst data sets over time and I’m also doing a long term growth sample. I’ll explain in my next post.

Here is my setup:

  1. Put 9ml of DI, DDW, and D2O YPD in 3 test tubes respectively (each in it’s own).
  2. Add 1ml of each yeast sample from the starter cultures.
  3. Starting at t=0 remove 400ul and put that in a semi-micro cuvette for nanodrop analysis. Do this every 60min.
  4. Incubate the cultures at 30C and 150rpm

Simple!

D2O adaptation, Water Type Effects on Organism Growth, Yeast

Starter Cultures Try 3!

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Gosh this yeast is tough to get started. It just can’t get going in liquid YPD. I finally got a few colonies to appear after 2 days on YPD plates. I inoculated a few of those colonies in some new liquid YPD (DI, DDW, and D2O) so hopefully tomorrow I’ll be cooking meth! LOL, just kidding… or am I?

I wonder if meth with D’s instead of H’s would affect the high you get… hmmmm….

D2O adaptation, Microorganisms, Water Type Effects on Organism Growth, Yeast

Starter Cultures part 2

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Yesterday’s setup didn’t work so well. I’m not sure what prevents the yeast from growing. I’ll have to make a glycerol stock of some yeast colonies so I have a supply of always working yeast. Anyways, today I made a bunch of new starter batches following the same protocol from yesterday. Crossing my fingers.

D2O adaptation, Microorganisms, Water Type Effects on Organism Growth, Yeast

YPD setup and Starter Culture prep

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This is going to be a hyper detailed post because I’m unable to work in the lab today (soccer injury) and so Steve will be filling in. So pardon me while I write the tiniest of details about the lab so Steve can access everything I need him to:

Making YPD:

  1. Supplies:
    1. You will need 3 beakers. The beakers are stored above the autoclave and there may be more in the cabinet below and to the right of the sink. You’ll want beakers that can hold well over 100ml because when you stir you may spill some.
    2. You will also need 3 bottles with caps for long term storage of the prepared YPD. Those are also above the autoclave.
    3. While you are here, the aluminum foil is in the drawer under the autclave, you’ll need some.
    4. The hotplate stirrer is in the front of the lab near the sink and microcentrifuge.
    5. YPD broth mixture is on the wet lab bench with all the powder chemicals right behind Kiney’s computer.
    6. The scale is in the front of the lab next to the PCR machine, and there are mini weigh boats around there too (aolong with regular sized weigh boats).
    7. Scoops and scoopulas are near the sink in the back. There are two cups one is labeled for dirty scoops and the other for clean. You’ll need a clean one.
    8. Medium gloves are in the top left most drawer opposite the autoclave (and under the seed growth station).
    9. Syringes and syringe filters are in the right most lower cabinet opposite the Kiney computer. There are some really big syringes (60ml I think) and filters. Use 1 filter per squeeze.
    10. Stir bars are magnetized to the bench right about eye level near the powder chemicals. You’ll need three, and you might as well grab a magnet too so you can get the stirbars back.
    11. New bottles of DDW are kept in the desicator next to Nadia’s bench. New bottles of D2O are kept next to the powder chemicals. You will need one of each.
    12. Sigma DI bottled water is above the seed growth station. You will need 100ml of this.
    13. The autopipetter is somewhere in the lab. That thing gets around quite a bit. It is either somewhere around Nadia’s bench, somewhere in the front of the lab (maybe in it’s holder near the pipette tips), or near my bench hanging above or near the laptop. Tips are in the front of the lab next to the peeper PC.
    14. Sharpie – these things are everywhere in the lab. If you can’t find one there should be like 3 sitting on my bench.
  2. Measure the amount of DDW and D2O in each bottle and put in beakers. Cover with aluminum foil. And measure 100ml of DI water and put into the third beaker.
  3. Calculate the amount of YPD needed based on what it says on the YPD bottle. From this post here, it looks like you need 5g per 100ml of water. And the D2O should yield about 90ml of water.
  4. Add the ypd to the beakers with water (and make sure you keep track of which beaker has which water) and one by one, using the stirrer, mix until dissolved.
  5. Filter this water into the bottles. It takes kinda a while to get full dissolving so while one is mixing you can syringe and filter an already stirred water into a bottle.
  6. Label the water type.

You’ve just made YPD in different water types! Yay! Ok now to make the starter cultures.

  1. Gather supplies:
    1. yeast – in the fridge on the right side (and maybe near the bottom) is a rack of PCR tubes with yeast starters in it. It should say yeast on the caps, but it may be hard to read.
    2. test tubes – are above the autoclave in a blue test tube holder. There should be 2 holders and one should be empty. You’ll need 3 test tubes.
    3. prepared ypd – you just made this!
    4. disposable inoculation loops – these are in the drawer under the laptop. There are two colors, green and blue and each is a different size. I can’t remember which one is smaller, but you’ll need the smaller one because the larger one won’t fit in the PCR tube.
    5. ypd plates – might as well streak some yeast while we’re at it. These are on the top most part of the bench above the seed growth station. Take out one or two if you please.
    6. aluminum foil – again, since you just used this it should be accessible to you.
    7. incubator/shaker – in the chase near the hole in the lab, where the banana picture was made
  2. Put 10ml of each water type into a test tube and label it. Sharpies abound on my bench so you should be able to find one.
  3. Use the inoculating loop to inoculate the yeast in the liquid media. One loop per tube and swoosh around! Fun right?! (BTW, the ?! combo is called an interrobang and there is an interesting wikipedia article on this.)
  4. With the ypd plate, streak some cells from the starter tube onto here. And since there will be a lot of liquid left in the tube, I like to just poor the tube onto another ypd plate to ensure something starts growing. Paranoia is a bitch.
  5. Bring your new colonies to the incubator, make sure the incubator is set for 30C and about 100rpm. According to this post I set it for 125rpm because I can.

Yay! You have made fire! Now just leave it til tomorrow when hopefully I can return to do the next phase of experiments.

Ligation, Molecular Biology, Shotgun DNA Mapping

3-piece ligation: BpALS, EpBR, 5′-bio/int-bio adapter

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A few days ago I did a 3-piece ligation that didn’t turn out so well. Today’s ligation is more like the traditional ligation that I was supposed to do with the exception that I’m using the pALS anchor instead of the pRL anchor.

I mentioned earlier, that BpALS (pALS anchor digested with BstXI) is better overall because it is longer and makes the optical tweezer operations a little easier and better.

pBR322 can be digested with EarI or SapI for use in our construct for DNA unzipping. I haven’t done any studies that show which method produces better results in terms of final yield, but I’m pretty convinced SapI will because you only have to perform one gel extraction. If you digest pBR with EarI you get two linear fragments. And according to separate analysis by Koch and myself the overhang that we need for the unzipping construct ligation is the longer piece (in the gel image linked above it is the brighter top band).

The benefit to following this method is you can perform a three piece ligation (essentially ligating the entire construct together in one shot) because the EpBR piece (pBR322 digested with EarI) can only ligate to the adapter, and the BpALS/BpRL piece can only ligate to the other end of the adapter (both adapters that I’ve been using work the same way).

And so today I’m doing the proper three-piece ligation reaction. The reaction setup and method can be seen in the table below: