DNA preps from S2, S7, A2, A7

I did DNA preps this afternoon on all 4 lines of anemones. I did all 4 because of Liz's email that suggested doing them in parallel for consistency between apos and syms. DNA is in the cell bio lab freezer in the yellow rack with our other samples. (What happened to our freezer box?)

I did not do any DNA preps from water, since I wasn't sure which water we would want to look at. I assume it would be water from the tanks, and not the water from the fingerbowls, right?

Frances

Anyone up for lab this afternoon?

I'm in the lab...I'm not sure who posted the message earlier about possibly doing stuff this afternoon, but if you're still free, give me a call or come find me here. I have some anemone feeding/caretaking to do, so I'll start with that and see if anyone shows up for DNA extractions.
Frances

Ok guys,

So given all these things that we have to re-do/do in a different way... when is this going to happen!? I can do things this afternoon after 12, but I would prefer not to do things alone. I think we should definitely have a meeting with everyone present to re-organize and re-evaluate what it is we need to do from here.

Ok, also, cell phones work too - so if you think calling may be easier. Let's try and meet this afternoon and get crackin' on some work tho!

Umm un Problemo? with Culture-Independent Protocol

Hello All,

Eric and I have come across a problem with what we have been doing so far:
1) We did not do a clonal extraction of the APO colonies!!! for the complete bacterial assay for the culture independent studies.
-- clearly this is a problem, since we need to compare the bacteria of the Apo and Sym guys~
---- Plan of action. Do a DNA extraction of the Apo (2 and 7). The protocol will be outlined in the NEW lab notebook for the independent studies!! This requires sonicating 3 or 4 anemones in 150ul of 2X TE and then following the lysis protocol in the notebook. After the lysis is completed, the DNA extraction can be completed. The kit is on our lab bench and we follow the protocol for tissue extraction (I think?) I'll write down the correct page number in the notebook.
------- After the DNA extraction is complete, we will need to Run a PCR..... AFTER the PCR is done DO NOT visualize it on a Gel before running the PCR clean up kit. The cleaning can not be done once the dye has been added to the PCR product, unless you run a gel purification kit, which requires cleaning the chunks of gel with bands.
---------After the PCR clean up do not visualize... or only run a small amount on gel because we need the PCR product to put into the competent cells/plasmids to be sent to WASHU for sequencing

2) Since the PCR product from the Sym clones/H2O was visualized on a gel, the PCR clean up kit wont work, see above for explanation
---Eric and I did not do the PCR clean up kit this afternoon b/c of such. I think that we will put the Pcr stuff back into the freezer, but they already have buffer in them, so its probs ruined.... well ruined in the sense that we cant run it on a gel and excise gel fragments.

3) WASHU went home. We will have to call back tomorrow. Also. what exactly is it that we are asking them.

LOVE, A and E

Hey guys,

Ok, so last night Reid and I ran a gel of the PCR products that were isolated (used pos. and neg. controls and a ladder). The gel is printed and attached to one of Reid's random sheets of paper on the lab table with the protocol for how we loaded the gel also written down.

We added way more ladder than we were supposed to and so the ladder just looks like a huge streak, but we can still ascertain some important things from the other lanes... mainly that we have isolated some DNA, which is good. And also that we have a spot in the neg. control lane which we're hoping is just left over primers from PCR. We did not run PCR cleanup on the products before loading into the gel, but we do have 10 uL of PCR products remaining that need to be "cleaned up".

SO, if anyone, I think Eric it sounded like you had time, could come in this afternoon and run the PCR clean-up protocol on the remaining PCR products (in freezer in Liz's lab) that would be wonderful. The kit and protocol are on our lab bench. If you're able to do this, please post ASAP or directly call me or Reid. Ok, thanks guys, hope this helps a little bit.

We also wrote notes in the lab notebook about what we accomplished last night. And by lab notebook, I again mean random piece of paper. We really need a culture indep. notebook!

Just so that we have it here...

Bacterial Colony PCR Procedures:

1. Draw grid on clean LB-amp or LB-carb plate (~28 sectors).
2. Select colonies to pick. Streak portion of colony to numbered sector and place the remainder in a correspondingly numbered PCR tube with 50uL of lysis buffer
3. Heat at 95 degrees for 10 minutes. ** This can be done in PCR machine.
4. Spin lysis solution on high for 10 minutes to pellet cellular debris, and remove 4uL for PCR

Lysis Buffer:
1% Triton X-100
20mM Tris*Cl pH 8.0
2mM EDTA pH 8.0

----

To my understanding, if we follow this procedure Ayanna and I shouldn't pick colonies tomorrow morning since we'd have to wait for the PCR kit to come... right? Also, do we have all the ingredients for the lysis buffer solution? Everything sounds familiar to me (except for the Triton X-100), so I assume that we do. That reminds me...do we have some sort of finalized-ish list of stuff that we need so that Liz can order it all for us? We might have to work on that as a group sometime soon.

Since it looks like I won't be doing anything for lab tomorrow now...is there anything that you guys want me to do tomorrow afternoon? I'm busy until 4, but I can do something afterwards (though I will be busy at night). I'm also free from 11:30 to 1.

Also, it'd be great if the people who worked on the PCR would blog about what was accomplished in lab today. I know that we diluted the necessary primers and created a gel for PCR. Did anything else happen after I left lab? I'm also still a bit confused on what needs to be accomplished by Friday. What exactly are we extracting the DNA from? What do we need to have done so that we can send off our stuff to be sequenced at WashU? Sorry if I sound a bit clueless about this part of the lab...but well I kind of am clueless lol.

- Eric

colony PCR info

Reid and I just talked to Liz about doing colony PCR on the bacteria currently growing on our plates. We found a protocol (http://www.cbs.umn.edu/labs/perry/Protocols/bactPCR.html) that Liz thought would work--basically you suspend a colony in lysis buffer, heat it for a while, spin it down, and use the supernatant as your PCR template.

Since lysis is more reliable on cells that haven't reached stationary phase, we will probably want to have fresh overnight colonies to use for this--instead of liquid cultures, we'll just grow them on agar.

We have also asked Liz to order more of those pre-mixed PCR kits for us. I don't think we/Amy's lab have enough tubes left to do all the colonies we talked about doing (~3 of each type from each anemone line), so we might have to wait to pick colonies until we know that the new PCR kit is on the way.

~Frances

Figures from Incubation of Sonicated Anemones in Sterile Sea Water

Figure 1. Sonicated Clone S7 1:10 dilution (symbiotic, 7) plated onto GASWA plates, no selectivity. Clone S7 anemones were sterilized and reacclimated in sterile sea water overnight before sonification. Plates were incubated and grown overnight at 30 C.

Figure 1a. Sonicated Clone S7 1:100 dilution (symbiotic, 7) plated onto GASWA plates, no selectivity. Clone S7 anemones were sterilized and reacclimated in sterile sea water overnight before sonification. Plates were incubated and grown overnight at 30 C.

Figure 2. Sonicated Clone A7 1:10 dilution (aposymbiotic, 7) plated onto GASWA plates, no selectivity. Clone A7 anemones were sterilized and reacclimated in sterile sea water overnight before sonification. Plates were incubated and grown overnight at 30 C.

Figure 3. Sonicated Clone S2 1:10 dilution (symbiotic, 2 plated onto GASWA plates, no selectivity. Clone S2 anemones were sterilized and reacclimated in sterile sea water overnight before sonification. Plates were incubated and grown overnight at 30 C.


Figure 4. Sonicated Clone A2 1:10 dilution (aposymbiotic, 2) plated onto GASWA plates, no selectivity. Clone A2 anemones were sterilized and reacclimated in sterile sea water overnight before sonification. Plates were incubated and grown overnight at 30 C.