Friday, August 26

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Dr Liz working alone in lab on Friday

The cell cultures in LB broth + amp that had been shaking in the incubator overnight were removed to the cold room about 5:00 pm. This amounted to about 17 hours, and we had set the incubator low - 31.5 degrees C. So I expect they were still in log growth phase.

Purification Protocol that includes Phenol:Chloroform Step

I used the DNA Purification by Lysis protocol from the Handbook of Molecular Cloning, Vol 1, Chap 1. Jessica has input this protocol on the wiki- I have not found it yet. Please link this page to the protocol if you can, somebody.

Essentials AND Variations of the Basic Protocol

There are a couple of modifications and special points worth discussing about using this protocol, as opposed to using spin tubes in a kit.

First: Everything must be and was kept very cold. I even put a vortexer in the coldroom next to the microfuge. Hint: If you know you're going to be working in the cold room for any extended period of time, make sure you are wearing warm socks and shoes. I'd even recommend a wool hat. By the time I was done with these cold steps in the purification process, I felt like a human popsicle!

Also, almost all centrifugation steps were performed on a microfuge in the cold room, according to The Handbook's protocol, "at 12,000 rpm and 4 degrees C". The only exception were the gentle spins needed to separate the aqueous phases from the phenol and chloroform (or chloroform:isoamyl alcohol).

Second: The phenol:chloroform step of the purification process is labeled as optional. However, the authors of Handbook, as well as other molecular biologists who have posted information on the web about the phenol:chloroform extraction step seem to agree on the following point: Downstream reactions may not work as well if you do not do the phenol:chloroform step. In particular, restriction digestion steps may not work well. One solution to a slow restriction digestion is to simply overload the amount of the restriction enzyme in the reaction - as much as a 10-fold increase is used in cases requiring better results. According to NEB, this is a fairly standard procedure, when needed. As we live on donations, it seemed to make sense to just do the phenol chloroform step instead. More information, particularly safety information, about doing this step of the purification is found below in the "Phenol:Chloroform" section.

The Basic Purification By Lysis Protocol, using Solutions I, II and III

I began the purification by following the protocol mentioned above, taking a 1.5 ml aliquot from each cell culture sample (each originally contained ~3.0 ml), and putting the remainder back into the cold room. I used Solution I and II, already available in the fridge and freezer from earlier in the month. and mixed a fresh batch of Solution II, as recommended. I made roughly enough for the nine samples. As when mixing Solution II for the first time early in August, I used the 0.4 M NaOH stock solution that was available (as opposed to 10 M NaOH, which is recommended). The 10% SDS solution used today had been freshly made by Cheryl Brown within the last several weeks, and was a different batch than the SDS in the Solution II used when the same basic protocol was first used this summer -- to purify DNA from cell cultures seeded with single-colony clones picked from amp plates that were inoculated with cells which had been transformed by plasmids containing one of P, R or T -- the first 3 BioBrick parts our 2011 iGEM Team extracted from the Parts Plates.

An interesting observation was that there appeared to be significantly more of cellular material in the bottoms of all of the 3B tubes compared to the 4A tubes after the initial spin down. The same observation persisted after the addition of the 3 solutions, the vortexing and the accompanying spindown that pelleted the biologicals. One hopes that this will correlate in a positive way to the amount of plasmid DNA left in the supernatant solutions after the last centrifugation step before coming around to the homestrech, described below.

The next step was the phenol:chlorofrm step, which we will postpone just long enough to finish discussing the other, last steps in the purification protocol. The last steps are DNA precipitation by the addition of at least 2.5 volumes of ice-cold 95% ethanol. The DNA precipitation with 95% ethanol is followed by a spin-down to form the DNA pellet in the bottom of the tube (It's actually the salts that you are seeing), and a pouring off of the supernatant, which should be free of plasmid DNA. I had some serious doubt when I did not see the DNA precipitate when I added the ice-cod 95% ethanol, but my doubt was solely that the DNA had not precipitated. I didn't doubt that the DNA was somewhere in the tube, i.e. I was not worried that I had "lost" the DNA at some earlier stage. Further, the spin down step did not produce a clearly visible pellet of DNA at the bottom of each tube. I saw what appeared to be a colorless, almost grainy-looking sandy sludge on the wall near the bottom of the tube where one would normally find the pellet. Now there seem to be a couple of opposing possibilities here. Perhaps my DNA got SO clean during the phenol:chloroform step that the pellet is hardly visible because it is relatively free of salts. The other possibility might be that I did not add enough ethanol to precipitate the DNA out of the quantity of solution that I had in the tube.

Saving certain byproducts that are normally discarded as a failsafe measure

Because of this doubt I employed a failsafe procedure that you might always consider doing if you are not certain that your work is going as it should. Namely, after the addition of 95% ethanol for precipitating the DNA and centrifuging the samples to pellet the DNA, I saved the supernatant that would normally be poured off and discarded. For these particular samples, they are labeled with the corresponding tube's name, followed by the "prime" symbol, which is just a single apostrophe " ' ".

I finished the procedure by performing the last step - adding 70% ethanol as a wash, gentle vortexing or inverting the tubes, centrifugation for 5-6 seconds and this time discarding the supernatant. I tried to be very careful not to wick away any of the clear "sludge"-like material that appeared in my tubes. According to the Handbook, one should be very careful when pouring off of this last ethanol wash, as one can accidently pour away the often-loose pellet of DNA at the bottom of the tube. See the "Finding Your Pellet after Centrifugation" section below.

The tubes containing the final product after the 70% ethanol wash are labeled without the primes. I did not elute the DNA in TE or water - I just left the DNA in the tubes, as my understanding is that this DNA is so stable in plasmid form that it does not even need refrigeration. I did put these tubes into a rack and placed it on the bottom shelf of the fridge, left side.

Warning Notes About Phenol

There are some important safety considerations you must know before opening a bottle of phenol. It is a DANGEROUS substance. Wear gloves and long sleeves and BE CAREFUL. It can burn your skin quickly, even in small quantities. It cause irreversible liver damage. And if that's not enough to scare you into your lab coats, gloves, googles and full footgear, how about this: IT CAN KILL YOU.

WORK UNDER THE HOOD AS MUCH AS IS FEASIBLE. If you accidently splash even a small amount on your skin. flush with lots and lots of water and GET TO A DOCTOR. If a significant amount somehow comes into contact with your skin then it is recommended that you first rinse with PEG 400, then rinse immediately with lots of cold water and GO TO THE HOSPITAL or CALL 911. The phenol soaks through your tissue, gets into your bloodstream and causes permanent liver damage. The burns are so bad they may require the burn victim to undergo skin graphting in order to repair the damage.

Back to the "Optional Phenol:Chloroform Step(s)

So, where were we? After washing the cell sample with Solutions I, II and III, vortexing andspinning down, the pellet that remains after the spin-cycle is cellular material and some proteins. The supernatant is poured into fresh, labeled tubes. The cell crud and tubes can be disposed of in the Hazardous Waste receptacle at the front of the room..

So, I mixed buffered (pH = 8.0) Phenol in equal parts to Chloroform -Isoamyl Alcohol, and then added to each tube of supernatant product from the previous step a volume of Phenol:Chloroform approximating the volume of the supernatant. This is gently but thoroughly vortexed. Imagine each molecule of cellular material still left -- mostly protein -- needs to be "captured" by phenol molecules and pulled doen into the phenol bottom phase during the centrifugation step, just moments away. Imagine also the action of the Chloroform. You see, we will use Chloroform alone as the final wash to remove the Phenol still adhereing to the DNA. Chloroform helps the Phenol "slide" off the DNA. Think about soap. It makes water wetter. Well Choroform makes Phenol slicker, and this property is responsile also for how Chloroform prevents shearing of DNA when the Phenol is bound to proteins that may be tangled up in some way to the DNA that the Phenol is going to wash. The Chloroform makes it easier for the Phenol, now attached to a protein, perhaps, toslide away from the DNA during the centrifugation step, while still adhering to the proteins the Phenol is designed to pull away from the DNA.

After centrifuging this supernatant with DNA and Phenol:chloroform solution, the heavier phenol chloroform will sink to the bottm, the aqueous "upper phase" contains the solubilized DNA, and there is a line where the two phases -- top and bottom - meet. That line is mostly proteins. After spinning the tubes, I observed very thin or virtually no line between the aqueous and phenol phases. So I carefully pulled off the top phase with a pipet set at about 100ul (or less as I got down close to the interface between the phases.

This was undoubtedly the most challenging procedure involved in this process. It was difficult to see through the dirty hood glass, my own dirty glasses, and also to get the tube up to a decent height, given the limitations of my arm length!.

I read a helpful hint that I then recalled using years ago -- it can be easier to suck away the lower phase, then recover the upper phase the is left.

Since the advice is to do the phenol wash as many times as needed to get that protein interface layer to disappear, I felt ok about only doing one phenol:chloroform procedure. Being unhappy with the amount of DNA that I saw must have been left behind in that portion of the aqueous solution that I could not risk taking with the pipet without also risking sucking up some of the proteins also (less DNA that is pure is better than more DNA that is dirty), I decided to do a quick extra step called "washing the upper phase." I added 100ul of nuclease-free water to each tube when I added the chloroform:Isoamyl alcohol solvent that would clean remaining phenol off the DNA. Then I vortexed the mixture, thoroughly but gently, so as not to break the plasmids. That gave me a larger amount of aqueous upper phase to shoot for, so leaving behind the same amount of fluid did not leave behind the same amount of DNA, because more DNA wassucked up since a greater volume of the upper phase could be sucked up with the pipet. I felt like that had been a good decidion.

Then is was on to the DNA precipitation steps, using the 95% ethanol, following that with a wash using 70% ethanol. Again, I may not have added enough ethanol, ul for ul, to the tubes, and that could explain why the DNA did not "snow out" of the solution. Adding that extra 100ul of water during the last wash with chloroform:Isoamyl alcohol cause the volume to go up significantly. I think I did not properly take this into account when adding the ethanol.

But I can try again on that, having saved the supernatant.

Question for the day: What is PEG?

Did not get to the "Gel Running" Stage

I was working arounddoing the purifications by hand, which left no energy for checking the reults for DNA. We should do that Tuesday, Aug 30.

Growing up a cell culture seeded by a single colony derived from a cloned cell

After the P, R and T parts were eluted by Patrick and other team members, they were used to clone competent cells, which were then plated on agarose +amp plates. Single colonies were chosen from the plates after incubation, and were grown up in amp-rich LB broth in the warm shaker. One thing we have learned since then is that parafilm should not be used to secure the lids of these tubes, as they were specially designed for shaking a cell culture overnight - the idea is to get air in there. We were concerned about the culture sloshing out of the tubes, because the lids seemed loose. We understood only that air is required for cell growth, but thought sufficient air would be in the tube if we filled each tube with cell culture taking up no more than 1/3 of the tube's volume. This air deficit might explain some things, including why there was no DNA on gels run after purification of the plasmid DNA from those cell cultures. Is it possible that there was not ample air for these cells to grow and to manufacture lots of plasmids at the same time? Hard to say.

What we can say is we DID NOT make this error again when the 9 cell cultures were put in the incubator on Thursday night, August 25.