Allen Cai's notebook

8/19 Since we were missing the enzyme Pvu I for awhile, I stopped cloning the fusion proteins for awhile. Now though, there is more Pvu I, so back to making more fusion proteins! I set up 4 liquid cultures of the 11 fusions I had. Ryan had some liquid cultures set up also, and I will help him mini prep those tomorrow.

8/18 FINALLY I have movies to take! It took like almost one month for these PI3K mutant cells with our fusion protein to grow. Not all of them grew though, only two more strains. HO 46 - 90 cells --> 9.0x10^5 cells/ml HO 2 - 30 cells --> 3.0x10^5 cells/ml HO 285 - 116 cells --> 1.16x10^6 cells/ml HO 349 - 313 cells --> 3.13x10^6 cells/ml

HO 46 - 111.1ul --> 888.9ul HO 2 - 333.3ul --> 666.7ul HO 285 - 86.2ul --> 913.8ul HO 349 - 32ul --> 968ul

HO 285 - looks like no red fluorescence, but not sure HO 349 - looks like no red fluorescence, but not sure

8/17 Oliver's back, and he sees our cells. The media looks too concentrated, so I replaced all the media of the plate. Oliver hope this will allow it to grow, but he says some look dead. :( Ryan sucked out the media from all the plates and I added 12ml of K5 media + hygromycin to all the plates. When replacing media, we found three strains contaminated, and one strain that was ready to be frozen down.

8/14 One strain of cells actually grew! HO 349. It took forever to grow though, only about one month. I froze this strain down today.

8/12 Mini prep of all the fusion proteins in destination vector. After mini prep, I sent them off for sequencing. Since the efficiency is so high from doing gateway LR, one of the two samples should be a positive.

8/11 HO 46 - 272 cells --> 2.72x10^6 cells/ml HO 2 - 35 cells --> 3.5x10^5 cells/ml HO 293 - 40 cells --> 4.0x10^5 cells/ml HO 294 - 53 cells --> 5.3x10^5 cells/ml

HO 46 - 36.8ul --> 963.2ul HO 2 - 285.7ul --> 714.3ul HO 293 - 350ul --> 750ul HO 294 - 188.7ul --> 811.3ul

I set up liquid cultures for the Gateway LR reactions we did yesterday. I set up two liquid cultures of each fusion protein, because Gateway LR reaction has a very high efficiency rate, because it only cuts and LR clonase only cuts, swaps the two pieces, and then ligates the pieces in place. Basically, if it doesn't recognize any restriction sites, then the LR clonase will not cut in the first place.

8/10 Gateway LR reaction

We do gateway LR reaction to get our fusion proteins into a destination vector, so when we transform our proteins into dicty the plasmid will have the genes necessary to survive inside of dicty and replicate. The destination vector has an RFP attached right at the end of where the fusion protein ends, so when transcription happens inside of dicty, the RFP will mark our protein. There are two catalytic domains that have a stop codon at the end of the fusion proteins, so if we attach an RFP in the back, it will definitely not make the RFP. To solve this problem, we have a different destination vector in which there is an seqence for RFP to be attached in front of our fusion protein.This means the first thing dicty would transcribe would be the RFP and then our fusion protein. This way we can still at least see an red fluorescence. We want to be able to see the RFP under a fluorescent microscope, so if we see the RFP we will know that our protein has been made inside of the dicty. Sometimes we may not see the RFP, because the RFP may be too little to be detected with the fluorescent microscope. If we don't see the RFP, it doesn't mean that the protein is not made from our plasmid. It should still be made, because only the cells that took in the plasmid would survive in the presence of hygromycin. All of our cells have our protein, we know that for sure, but not all of them express RFP. Dicty may just not like that RFP protein when combined with our fusion protein, so that may be another reason why it may not express the red fluorescence.

I looked on the list of fusion proteins on our charts, and moved everything that wasn't on the destination vector chart. That was 15 fusion proteins I moved onto destination vector. All these 15 fusion proteins went into the pHO 203, the destination vector with the RFP at the end of the fusion protein sequence. fusion protein    destination vector pHO 352     -->       pHO 203 pHO 353     -->       pHO 203 pHO 354     -->       pHO 203 pHO 359     -->       pHO 203 pHO 372     -->       pHO 203 pHO 373     -->       pHO 203 pHO 374     -->       pHO 203 pHO 383     -->       pHO 203 pHO 384     -->       pHO 203 pHO 385     -->       pHO 203 pHO 386     -->       pHO 203 pHO 387     -->       pHO 203 pHO 388     -->       pHO 203 pHO 389     -->       pHO 203 pHO 390     -->       pHO 203

8/6 MORE MOVIES!! HO 46 - 82 cells - 8.2x10^5 cells/ml HO 2 - 66 cells - 6.6x10^5 cells/ml HO 293 - 306 cells - 3.06x10^6 cells/ml HO 294 - 255 cells - 2.55x10^6 cells/ml HO 192 - 129 cells - 1.29x10^6 cells/ml HO 196 - 62 cells - 6.2x10^5 cells/ml HO 197 - 57 cells - 5.7x10^5 cells/ml HO 198 - 75 cells - 7.5x10^5 cells/ml HO 200 - 44 cells - 4.4x10^5 cells/ml HO 202 - 70 cells - 7.0x10^5 cells/ml

HO 46 - 122ul --> 878ul HO 2 - 151.5ul --> 848.5ul HO 293 - 32.7ul --> 967.3ul HO 294 - 39.2ul --> 960.8ul HO 192 - 77.5ul --> 922.5ul HO 196 - 161.3ul --> 838.7ul HO 197 - 175.4ul --> 824.6ul HO 198 - 133.3ul --> 866.7ul HO 200 - 227.3ul --> 772.7ul HO 202 - 142.9ul --> 857.1ul

I sent in more mini prep ligations in for sequencing to check if the ligations worked.

8/4 HO 46 - 126 cells HO 2 - 58 cells HO 293 - 337 cells HO 294 - 332 cells We want to count the cells, so we can get exactly 10^5 cells of each strain into the 12-well plates. To find the concentration of our plates, we multiply the number of cells we count by 10^4 to get the concentration. HO 46 - 1.26x10^6 cells/ml HO 2 - 5.8x10^5 cells/ml HO 293 - 3.37x10^6 cells/ml HO 294 - 3.32x10^6 cells/ml

To find how much ul we take out from each tube and put it into the well, we use the formula C1V1=C2V2 C1 = the concentration of our sample V1 = how much we want to take out from our tube C2 = the concentration of cells we want to take out (10^5 cells) V2 = we fill the rest of the well with K5 media, and we want a total of 1000ul of liquid in the well V1             V2 HO 46 - 79.4ul --> 920.6ul HO 2 - 172.4ul -->827.6ul HO 293 - 29.7ul --> 970.3ul HO 294 - 30.1ul --> 969.9ul

I was not sure of if HO 293 or HO 294 was RFP fluorescent.

I sent in stuff for sequencing from our mini preps of ligations.

8/3 We have to count cells so we get 10^5 into each plate. HO 2 5.9x10^5 cells/ml HO 293 1.85x10^6 cells/ml HO 294 2.19x10^6 cells/ml

HO 2 16.9ul HO 293 5.4ul HO 294 4.6ul

I set up 48 ligation liquid cultures. I did 4 of each ligation, so I only did 12 plates for now, since i have so much.

7/31 After tracking all our movies with Angi's program, it is now time to analyze the data we got. From the results files, we generated histograms, comparing our WT strains with the strains that contain our fused proteins. We looked at the p-values for each histogram to determine if there was a difference at all. The p-value tells us how different the graph or how similar the graph is. If the p-value of a graph is 1.00, that means the two strains are 100% similar, and if the p-value of the graph is 0.00, that means the graph is 0% similar (100% different). When comparing our WT strain and strain that contains our fused protein, we want the p-value to be as low as possible, so there is a difference in the strain. We are comparing the straightness the cell moves and the total speed of the cells. We are really more interested in the speed of the cell, but if a strain is able to move relatively straight with the protein we introduce to them, it can help a cell move to a certain location faster, because it can move straight. Moving in a straight line is much faster than moving in many many circles, before reaching the same place. We also generated histograms of the same strains, but on different days, to see how similar the strains were on different days. We want this p-value to be as high as possible, so it shows our experiments can be repeated. When looking at these histograms though, the p-values weren't too high and around the 0.50 area. This tells us that dicty varies from day to day. It never seems to be the same on different days. This info tells us that we MUST have the WT strain, HO 46, every time we do microscopy, otherwise, we can't tell if the cells are just moving crazy fast because of our protein or it's just moving fast that day. I found out that HO 200 and HO 202 had the biggest changes in speed and straightness. After making the histograms, we watch the movies over and over to see if we find something else interesting that data can't detect. I saw that HO 192 was able to drag another cell along and pull it somewhere else, like how Wendel showed us those algae pulling microscopic beads. Maybe these proteins give super strength?

7/29 Since I forgot the make a control plate of fruiding bodies, HO 46, I have to redo all my fruiting body assays. Without the control, I have nothing to compare my strains with. Since I was setting up fruiting bodies, I also set up cells for microscopy, since my first videos were unuseable data.

Counting HO 46 - 105 cells      1.05 X 10^6 cells/mL HO 192 - 54 cells      5.4 X 10^5 cells/mL HO 196 - 42 cells      4.2 X 10^5 cells/mL HO 197 - 40 cells      4.0 X 10^5 cells/mL HO 198 - 58 cells      5.8 X 10^5 cells/mL HO 200 - 55 cells      5.5 X 10^5 cells/mL HO 202 - 40 cells      4.0 X 10^5 cells/mL

After getting concentrations of our cells, we need 20000 cells/mL for fruiting bodies and for microscopy we need 4 X 10^4 cells/mL (since I'm leaving overnight, dicty divides, so we don't want 10^5 cells).

For fruiting body HO 46 - 19.1 ul HO 192 - 37.1 ul HO 196 - 47.6 ul HO 197 - 50 ul HO 198 - 34.5 ul HO 200 - 36.4 ul HO 202 - 50 ul

For microscopy HO 46 - 38.1 ul HO 192 - 74.1 ul HO 196 - 95.2 ul HO 197 - 100 ul HO 198 - 69 ul HO 200 - 72.7 ul HO 202 - 100 ul

I did mini preps on the newly formed fusion protein with the catalytic domains of pHO 352, 353, 354.

7/28 I did the mini preps of the old fusion proteins and the Gateway LR reactions. With every fusion protein mini prep, we test digest the plasmid to see if it contains our proteins. A test digest means that we cut the plasmid with an enzyme. It doens't matter what enzyme it really is, but in our case its Pvu I. Our plasmid contains only one Pvu I site, so its ideal to use this enzyme. Since cutting with Pvu I linearizes our plasmid, we know anything that is bigger than 3k bp, will maybe contain our fusion protein. We run our digest on a gel and look at the gel. Anything that is 3k bp means that its just vector and we don't really care about only vector. If we find something that is bigger than 3k bp we send that sample in for sequencing. We have to check the sequence of the plasmid so we know that our fused protein is the correct sequence, before we move onto the Gateway LR reaction.

I set up liquid cultures of the newly ligated catalytic domains with my localization domains: 43-352, 353, 354, 45-352, 353, 354, 47-352, 353, 354, 48-352, 353, 354.

7/27 The localization domains that I'm in charge of are pHO 43, 45, 47, 48. Since Ryan Q purified more new catalytic domains, we have to ligate all those new catalytic domains with our localization domains. The new catalytic domains are pHO 352, 353, 354. We are always trying to find more catalytic domains we can test that can potentially make a better brake or accelerator. We have to do a lot of calculations to get exactly 2M fold excess of each piece. After letting the ligations sit for 30 minutes we have to get these newly created fusion protein plasmids into cells and grow them for mini preps. We transformed these plasmids into TG1 cells and plated it onto kan plates. The vector contains a kan resistance gene, so we know which colonies may contain our new fusion protein. The vector itself may also close up on itself, that's why we pick more than one colony in hopes we can get a band on a gel higher than 3k bp.

I picked only two colonies of each Gateway LR reactions i did yesterday, since the Gateway LR is so efficient. We can almost guarantee that the reaction worked, if there are colonies on the plates. After mini preping the liquid cultures we send it in for sequencing to make sure that the plasmid is athe correct sequence, since the next step is transforming this plasmid into dictyostelium. We don't want dicty to be making the wrong protein.

7/24 Microscopy Fluorescence HO 46 - no HO 192 - yes HO 196 - no HO 197 - yes HO 198 - no HO 200 - yes HO 202 - no

Did mini preps for the missing fusion proteins that is still missing on the big list of constructs, 43-60, 45-60, 45-133, 47-60, 48-60, 48-133. First number is a specific localization domain and the second number is a specific catalytic domain.

I set up liquid cultures of the Gateway LR reactions, so I can mini prep them tomorrow.

7/23 After doing ligations, to test if our ligations worked, we pick colonies from our plates and do mini preps on them. After doing mini preps, we cut it with the enzyme Pvu I and run it on a gel. We should only see one band, and if that band is 3000 bp long, then its only vector. If we see anything higher than 3000bp then we send that in for seqencing, and if the seqencing comes out correct, then it is ready for the next step. I did mini preps on 43-60, 47-60, 47-133, 45-60, 48-50, 48-66, 48-133. The first number is our localization domain, and the second number is our catalitic domains.

I also did Gateway LR reations today. This is the step after seqencing our fusion protein and its correct. Basically Gateway LR reations cuts out our fused proteins and paste it into a destination vector all in one step. The destination vector has all the requirements for the plasmid to survive when we put our plasmids inside of dicty. pHO 231             pHO 203             pHO 181 < destination vector pHO352              pHO 294             pHO 339 pHO 353             pHO 298             pHO 340 pHO 354             pHO 346 pHO 92              pHO 133 pHO 60 pHO 355 pHO 325 pHO 356

I set up more liquid cultures for the failed mini preps.

I set up 4x10 4 cells/ml for tomorrow's microscopy.

7/22 Mini preps on liquid cultures. Pvu I digested. Ran a gel.

7/21 Mini preps on liquid cultures. Digested with Pvu I. Ran a gel.

Get 1x10 5 cells for each strain for microscopy.

RFP HO 46 - not fluorescent HO 196 - not fluorescent HO 200 - fluorescent HO 202 - not fluorescent HO 192 - fluorescent HO 197 - fluorescent HO 198 - not fluorescent

Set up liquid cultures for pHO 43-60, 47-60, 133, 45-60,133.

7/20 Set up fruiting bodies for HO 192, 196, 197, 198, 200, 202.

Set up liquid cultures for pHo 48-50, 66, 92, 60, 133, 43-60, 133, 47-60.

7/17 Gel purified pHO 48 and pHO133.

Re did all the ligations for AB pieces and pHO 60 and pHO 133. Transformed the ligation into DH5a cells.

Ryan Q and I transformed 68 of our constructs into dicty cells.

Split the other transformed dicty I had.

7/16 Mini preps to get more plasmid for dicty transforemation. Also for Gateway LR reactions, and ligations. Digested ligation mini preps with Pvu I and ran a gel. Sent Gateway LR mini preps for sequence and positive looking bands on gel to sequencing.

Aar I digest on pHO 48 and pHO 133.

7/15 Mini prep on my Gateway LR.

Gel purified pHO 60 and pHO 44.

Ligation of all pHO 43, 44, 45, 46, 47, ALW 148 to pHO 60 and 133. Transformed into DH5a cells.

Set up many liquid cultures for out destination vector plasmid.

Froze our dicty transformation cells.

7/14 Mini preps on pHO 44-50, 46-50, ALW 148-66, 55, 99, PIPK, INP. Digested with PVU I. Ran a gel.

Added Hygromyosin to dicty transformation plates.

Redid Gateway LR reactions on the plates that showed nothing, pHO

Gel purified the pieces pHO 66 and pHO 133. I failed getting one piece from pHO 60 so I did Aar I digest on that again. I also did Aar I digest on pHO 44, because we almost have no more of that AB piece and we want to incorperate pHO 60 and 133 to these AB pieces.

7/13 Mini preps on pHO 48-66, 50, 92, 93, 97, pHO 46-50, 93, 97, pHO 44-50, 97.

Split our dicty cells.

Transformation of pHO 275 in to pHO 46.

Gateway LR reactions on pHO 292, 293, 294, 295, 296, 297, 298, 248 into the destination vector pHO 203. We also transformed PD and PD* into bacteria.

Aar I digest for pHO 60, 133.

Set up liquid cultures for pHO 44-50, 46-50, PIPK, INP, ALW 148-66, 55, 99.

7/12 Set liquid cultures for pHO 48-66, 50, 92, 93, 97, pHO 46-50, 93, 97, pHO 44-50, 97.

7/10 Mini prep on our Gateway LR plates. Digested with Hind III and Bgl II. Ran a gel.

Gel purification on ALW 150.

7/9 Mini preps on pHO 44-50, 46-50, 46-66, 46-93, 46-144, 44-97. Digested with Pvu I and ran a gel.

Set up Aar I digest for ALW 150.

Set up to make more fruiting bodies.

7/8 DBold textid mini preps on pHO 44-92, 50, pHO 46-93. Digested with Pvu I. Ran a gel.

Gel purification for pHO 50, 92, 93, 99

Gateway LR reactions for pHO 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 261, 248, 93, 48, 46, ALW 148, 150. Transformed gateway LR into DH5a cells. Plated the cells on CARB plates.

7/7 Added hygromyocin to our transformation of dicty to select for the ones that got out plasmid.

Set up liquid cultures for the constructs that were negative on the gel.

Did more Aar I digests for the pieces we ran out of.

Sent in the positives on the gel for sequencing.

7/6 We learned how to do dicty transformation. I transformed pHO 235, 269, 273, 275, 276, 27, 278, 279, and 280 into pHO 46 cells with G418 media.

We did mini preps on the 116 cultures today.

We did a digest with Pvu I.

We ran a gel.

7/5 I set up 116 liquid cultures. There were 29 plates, and I picked 4 colonies from each plate.

7/3 I did mini preps on the liquid cultures I set up yesterday.

I did gel purification for the pieces pHO 44, 46, and 48.

We did ligations, combining an AB piece to CD piece with a BC linker.

Transformation of our ligations into TG1 cells, and plated onto a KAN plate.

7/2 We set up plates to make fruiting bodies. We have to wait at least like 4 days for the actual fruiting bodies to form.

I did gel purifications. I had to purify the pieces pHO 55,92,48, and ALW 150.

I set up Aar I digests for pHO 44, 46, and 48.

I grew pHO 256 with the destination vector pHO 203 and pHO 50 in liquid culture overnight.

7/1 Mini preps of our plasmid transformation and Gateway LR reactions.

AarI We digested the plasmids from our mini prep of the plasmid transformation with Apa I. These are still parts that we want to test out. I had the pieces pHO 55, 92, 48, and ALW 150. We did PCR purification to remove the enzyme, Apa I, from the mix. After the PCR purification, we digested the sample with Aar I.

Gateway LR plasmids We are doing test digests on these samples to make sure that there are no mutants in the plasmids. My first gel came out weird, the whole top of the gel was missing. I think this is because that i placed the combs to low, and it therefore didn't create a well for my samples anymore. I redid my test digests, and ran another gel. The gel looked great!

6/30 We purified plasmid from our liquid cultures. After we did a digest, using the enzyme PVU I.

Digest: Water             5.3µl                             116.6µl Buffer 3          1µl                               22µl PVU I             0.2µl           x22               4.4µl BSA               0.5µl                             11µl DNA               3µl total             10µl                              154µl

We incubated digests at 37C.

We ran our digests on a gel at 130V.

There were positive results, and we are sending in pHO 43-92-2, pHO 43-92-3, pHO 43-92-15, pHO 43-92-19, and pHO 43-92-20. We set up the reaction that will be sent.

Water     5ul DNA       5ul 10ul

Today we set up more liquid cultures for mini preps tomorrow. It is for the Gateway LR reations, and plasmid transformation. I will take over Ryan Q's plate ALW 150. The plates we grew yesterday looked great. The Gateway LR reaction plates had many isolated colonies. The plasmid transformations looked like a lawn.

6/29 Today we did Gateway LR reactions. I had the constructs ALW 183, ALW 188, ALW 193, ALW 227, ALW 231, and ALW 235. The Gateway LR reactions is a cut and paste in one step. After the Gateway LR reation, we transformed it into plasmid. We also did transformations for parts we still needed that were on a plasmid, pHO 48. We used CARB plates for the ALW pieces. For the pHO 48 we used a CARB and Amp plate, because we don't know which resistance is on the plasmid.

Today we set up liquid cultures for our mini preps. I picked 20 colonies of pHO 43-92.

6/25 Today we ran gels of our digests. We loaded 1ul of dye into all of our digests.

Lane 1 - Ladder (can't be seen) Lane 2 - pHO 45-50-1 Lane 3 - pHO 45-50-2 Lane 4 - pHO 45-50-3 Lane 5 - pHO 45-50-4 Lane 6 - pHO 45-50-5 Lane 7 - pHO 45-50-6 Lane 8 - pHO 45-50-7 Lane 9 - pHO 45-50-8 Lane 10 - pHO 45-50-9 Lane 11 - pHO 45-50-10 Lane 12 - pHO 45-132-1 Lane 13 - pHO 45-132-2 Lane 14 - pHO 45-132-4 Lane 15 - pHO 45-132-5 Lane 16 - pHO 45-132-6 Lane 17 - pHO 45-132-7 Lane 18 - pHO 45-132-8 Lane 19 - pHO 45-132-9 Lane 20 - pHO 45-132-10 Lane 21 - pHO 45-55-1 Lane 22 - pHO 45-55-2 Lane 23 - pHO 45-55-3 Lane 24 - pHO 45-55-4 Lane 25 - pHO 45-55-5 Lane 26 - pHO 45-55-6 Lane 27 - pHO 45-55-7 Lane 28 - pHO 45-55-8 Lane 29 - pHO 45-55-9 Lane 30 - pHO 45-55-10

Dictyostelium

Our cells were 100% confluent again today, so we split the cells.

6/23 Today we purified plasmid from our mini preps. After that we did digests on our plasmid

Digest: Water             5.3µl                             116.6µl Buffer 3          1µl                               22µl PVU I             0.2µl           x22               4.4µl BSA               0.5µl                             11µl DNA               3µl total             10µl                              154µl

6/22 Mini Prep

The pHO 45-132 piece looked like it had one positive result, but looks somewhat aligned with the vector, since the gel ran a bit slanted. Oliver decided I should just do mini preps on the pHO 45-132 pieces again. This time we are picking 10 colonies, so our success rate is higher on getting a positive result. Today, I picked ten colonies and grew the cells overnight.

Dictyostelium

Our plate was truely 100% confluent today, just growing over the weekend. We split the cells again today.

6/19 Gels

Today we ran gels of the digests we did yesterday. We learned how to set up a 1% agarose gel (4g of agarose powder, 196ml buffer) and how to make penny surface tension gel (three pennies, two combs, melted agarose, etidium bromide). We poured 25µl of melted agarose into a tube and add just a coated pipette tip of etidium bromide to the agaraose. Then we poured it on the glass with three pennies under it and two combs (one on top and one in the middle). We put the gel in the electophoresis chamber, and loaded a 500kb ladder (I think) in lane one. I then loaded my 15 digests in the next 15 lanes. We let the gel run at 130V until the light blue color of the ladder reached the bottom.

Lane 1: Ladder Lane 2: pHO45-132-1 Lane 3: pHO45-132-2 Lane 4: pHO45-132-3 Lane 5: pHO45-132-4 Lane 6: pHO45-132-5 Lane 7: pHO45-144-1 Lane 8: pHO45-144-2 Lane 9: pHO45-144-3 Lane 10: pHO45-144-4 Lane 11: pHO45-144-5 Lane 12: pHO45-55-1 Lane 13: pHO45-55-2 Lane 14: pHO45-55-3 Lane 15: pHO45-55-4 Lane 16: pHO45-55-5

Some lanes are clearly bigger than the other, which means the ligation we did at school (combining four peices together) worked. The gel ran a bit slanted, so the last few lanes are unsure, but it looks positive.

Dictyostelium

Today, some parts of the plate was 100% confluent, while the other parts were 50% confluent. Oliver told us to split the cells anyways, because it was going to be the weekend, and we didn't want the cells to overgrow. Since we had less cells than the other groups, instead of removing ALL the media, we left 2ml of media of cells in the plate and added 10ml of new media.

6/18 Digests

Today we did digests of our plasmids. We cut the plasmids with the enzyme PVU I to linearize our plasmid. We made a master mix of all the ingredients, leaving out the DNA. This way we can pipette a lot less. We did these reactions in PCR tubes, because it was like a control digest (I think).

Digest: Water             5.3µl                             95.4µl Buffer 3          1µl                               18µl PVU I             0.2µl           x18               3.6µl BSA               0.5µl                             9µl DNA               3µl total             10µl                              126µl

We left the digest at room temeprature for two hours.

Dictyostelium

Our cells look great. The confluency of the cells of some parts are 60%, while other parts are less, like 30%.

6/17 Mini preps

Today, we did mini preps on the cells we grew overnight. We purified the plasmid into 50 µl of elution buffer. After purifying, we stored our samples in the freezer.

Dictyostelium

We observed our cells today, and the cells only looked 20% confluent. Our cells seemed to grow slow, or we just got too little cells to start with. Some areas looked 5% - 10% confluent.

6/16 Dictyostelium

We learned how to plate dicty cells from Oliver. After, Oliver split us up into groups of two, so we can plate our own dicty cells. My partner is Cathy, and the cells we plated will be our cells to use for the rest of the summer.

Mini Preps Since our gels from our first mini preps looked very confusing and terrible, Oliver decided we should pick five more colonies to see if we can get results. The pieces i have is pHO45-132, pHO 45-144, and pHO45-55. We picked five colonies from each plate and grew these cells in liquid culture overnight.