Ethan Chan's notebook

From 2009.igem.org

8/5 Today i had to set up for some movies. I was able to film 8 strains today. All of these strains are HO46. HO46,HO231, HO226, HO190, HO208, HO204, HO179, HO209.

I also prepared for the next day's movies. When we prepare for the next day's movies, we will need less cells because they will grow overnight. We will only need a concentration of 4x10^4 cells

8/6 In the morning, i took movies of the strains that i prepared yesterday. Then edna and I prepared some pten mutants fruiting bodies. These plates will take about a week to grow before we see any results. Refer to her notebook for strains we did. I split cells and then prepared for tomorrow's movies. The strains i prepared are HO46 HO213 HO226 HO190 HO208 HO204 HO179 HO209.

8/7 I split my dicty strains and also took video of the strains i prepared yesterday.

8/10 I split the cells in the morning, and then checked the pTEN transformation for confluency. I froze down some cells that were confluent by spinning them down and adding freezing media. In the afternoon, i did analysis from the movies i have taken. I generated histograms and celltracked the movies by using the matlab program Celltracker. Some movies were too dark, so i needed to photoshop the dark pTens videos. They were able to track more cells after i changed the light levels of the video.

8/11 I continued to photoshop the dark pten videos. I was also able to generate and track the files so that they can be ready for analysis. In the afternoon, i prepared cells for videos for the pTEN strains. I took video of HO12 HO319 HO318 HO317 HO229 and HO325.

8/12 We had a meeting in the morning so we couldn't do any labwork in the morning. In the afternoon, i recorded the second set of yesterdays movies. I recorded HO12 HO317 HO318 HO319 HO325 and HO229. I froze down HO348 and HO347.

8/13 I generated histograms for yesterday's movies. Delquin will help us do analysis on these movies in the afternoon. I also did fruiting bodies for 2 of my strains. (HO348 and HO347) After the fruiting body experiment, i was able to take videos of HO46 HO347 and HO348. After delquin looked over some of my results. we concluded that HO165 HO166 HO170 HO178 and HO319 were not significant and could be thrown away.

8/14 Today i will be doing some recording later on the day. i will mostly be working on my Wiki and on the computer. I will need to do my plasmid sheet and also finish up my studnet statements.

Untitled Post posted ‎‎Aug 4, 2009 3:31 PM‎‎ by Ethan Chan

7/20 Today we learned how to use the flourescence camera. this is needed in order to check if any of our constructs are being expressed in the cell. On our Destination vector, there is a RFP that is attached. If there are any flourescence of RFP, then we can be sure they are working. The microscope settings is 20x objective lens, 2 miliseconds exposure time for regular brightfield movies and 500 milisecs for the flourscence ones. We will take video for 15 seconds intervals for 10 minutes. The camera was very easy to use. we will now start to record videos.

7/21 All the video we been doing requires us to have it in a certain condition. the cells cant be unhappy or else its action might change. We have the cells at 1x10^5 cells per well. Before we record, we need to set them up for video. We need to wash the cells off the plates. Pull out 1ml, count them and then take out the appropriate amount. We bring up the volume to a total of 1ml with media. We incubate for 30 minutes, asperate it out, and replace with 2ml kk2 buffer. we then wait half an hour before starting the video. The videos came out pretty well.

Count the number of cells, times 10^4 and use that for the concentration. to calculate how much to pull out, take 1x10^5 cells/well and divide by the concentration.

We also freezed down the cells, using the DHSO freezing media. We are doing this just to keep a copy of each strain just incase we have contamination on our plates.

Mini-preps were done and digested with pvu1. Only 44-50 worked and will be sent for seqencing. 46-133, 44-133, 46-60 46-50 aqnd 44-60 will need to be redone.

7/22 We did Minipreps and Digest in the morning. Some worked and some once again failed. We might have to religate them because they have failed often. I also set up some cells for video assay. HO162 Ho12 Ho173 Ho179 Ho172. I also Transformed 9 of the strains into HO46.

7/23 I set up some movies in the morning. I believe i actually stopped recording the video because i was told i left the cells in the tube for too long. i stopped the recording and disposed it. I moved on to making fruiting bodies of ho190 and ho204. These plates will be growing within the next week. I wont be able to see any results until next week.

7/24 I set up some movies to record in the morning. HO172,ho173,ho179, ho165, ho178, ho 170, ho166, ho162, ho12. Edna ran the PVU1 digest after doing the miniprep. I ran the gel once i finished with the movies. I was doing the movies during the miniprep so she took over. I checkd our pten strains and froze down ho218, ho219, and ho220. These strains will have to be transferd into liquid nitrogen tomorrow. I replaced the media with HL5 + hygro.

More fruiting bodies are being prepared. I did HO209 Ho208 and ho46.

7/ 27 Made more places that are running low. i did fuiting bodoes on 2 of my strains. We also froze some strains down that were confluent.

7/28 Ligations for new peices. These peices are new peices that are being introduced into our project. they are more cataylic and localization. Since my A-B because is 46, i will need to ligate all of these C-D to my A-B peice.

I made more plasmids for our stock because some of the tubes were running low. We made more plasmids again because 2 of the plates didnt grow any colonies. I diluted the sample in 49ul of water, took out 1ul. added 10ul KCM (5x) and added 39ul of water. We needed to thaw TG1 cells out and then added 50ul of cells. Ice for 2-5 minutes, 42* C heat for 90 secs, and then back on ice for another 2-5 mins. We place 50ul on a Carb plate and let it grow overnight.

7/29 The results for yesterday's plates were good. the colonies grew on the plates so i could prepare the mini-prep

7/30. I did more mini-prep in the morning and by the time i was done, it was time for the picnic with Jbay.

7/31 absent, wasn't in the office

8/3 I did fruiting bodies for HO231, and HO226. These plates will have to sit for about a week before i get to see any results. I learned how to use the Matlab programs to analyze our results. combine: combineresults generate histogram: generatehistograms

Did some Transformations for the pten mutants. refer to Edna's notebook to see which strains were done

8/4 I split my cells in the morning because they are getting very confluent. These plates will have to be thrown out soon because we have analyzed them and made fruiting bodies. I combined all my videos and generated histograms for all my strains. i will be getting data off of these programs later on today.

I added hygromyosin to the transfermations we did yesterday.

Untitled Post posted ‎‎Jul 20, 2009 9:27 AM‎‎ by Ethan Chan

7/9 We did our Mini preps in the morning. Digested them with PVU1 in the afternoon. We started our Fruiting Body Assays. We started by doing calculations and making dilutions of the cells. We plated the cells in 2 different dilutions for each strain.

7/10 Mini preps for the LR reactions that were sucessful. We digested them with HindIII and BglI. We did a master mix of 33. 203-46 203-ALW148 203-261 203-285 203-283 203-290 203-alw150 181-93 203-284 203-287 203-281 181-97 203-48 203-291 203-280 203-282 203-248 203-289 203-288 203-281 We also split our dicty cells with media with hygromyosin. i observed that i found mold( contamination) in one of my plates. I made sure to throw it out so it doesnt keep growing. we send in the samples that looked good for sequencing for the LR reactions.

7/13 We did Mini preps today. We digested them with PVUI and ran a gel. The ones that look good will be sent for sequences. ransformation: i am transforming pho316,pho302, pho 270, pho266 into dicty. We learned how to use the electrophoration machine to creat pores. the plasmids will be able to go in.

7/14 We did transformation into dicty cells. We had to calculate how many cells to put into each plate. Mini-preps and PVU1 Digest in the afternoon 46-50 Alw184-55 44-50 ALW148-99 ALW148-66

7/15 Added hygro to transformation plates, from yesterday Freezed our cells in 500ul of freezing media. We needed to spin the cells down and then resuspend. pho226/ho165 pho234/ho170 pho243/ho178 pho238/ho173

7/16 edna and i started our pten transformations into dicty. we did 32 transformations for the Pten mutants. We will redo the ligations that have continued to fail. Those peices are pho46-50 and pho44-50. these plates will be grown overnight.

7/17 We added dead bacteria to yesterdays transformation and hygromyosin. we did transformations for another 31 strains. I assisted in Alex's mini preps and also Allen's Transformations.

7/2 to 7/8 posted ‎‎Jul 8, 2009 11:40 AM‎‎ by Ethan Chan

Gel Purification: we cut out the peices that we needed. My A-B peice was ALW 148, and the C-D pieces were pho144, pho50, pho60. For the Pho144, we needed to cut out the .5kb piece, pho50: 1kb, pho 60: 1.6kb peice and ALW: 1kb peice. The process of cutting off the gels was a long one. Sometimes we couldnt see the bad on the smaller sample(next to the ladder) so we had to estimate it.

Later that day, Allen helped me transform them.

7/3 With all the individual parts we have, we needed to ligate them all together. Instead of doing the ALW148, i was told to do the PHO46 (a-b) instead. We needed to calculate how much of the A-B peice we needed and also the C-D peice.

7/6 In the morning, we ran transformations for the Phos without a strain number. I proceeded for the minipreps that we grew overnight. I took charge of the Pho46s. I completed the mini-prep, did the digest with PVU1 and then ran a gel. I did get a few positive, and they were sent to another lab for sequencing. The ones that didnt work will have to be repeated.

We transformed the cells into dicty by shocking them using a machine. We plated them on media

7/7 In the morning, we had a lecture bout how to use the microscopes. The positives for yesterday's gels were sent to the lab for sequencing. 46-55-1,2 46-144-4 46-92-3 46-143-1,3 46-132-1,3 For the plates that went through transformation yesterday, we added (12ul) antibotics to the plates.

For the gel that had negatives yesterday,We poked single-colonies and then grew them overnight.

We made movies from Oliver's sample dictys using the microscope. The footage was taken every 15 seconds for 10 minutes. We ran the AAR1 for pho 50,92, 93, and 99. Used 10ug DNA in a 135ul reaction.

7/8 The cells that have grown overnight was taken out in the morning. I have continued to do the mini preps for Pho46-97, pho46-50 abd pho46-66. Each plate was poked 6 times for a total of 18 tubes. The digest is currently running, and i am about to run it on a gel in a few minutes.

Untitled Post posted ‎‎Jul 3, 2009 4:42 PM‎‎ by Allen Cai

7-1 Spun down all the cells down in the morning to start ourmini preps. We had 29 tubes in all. The tubes edna and i were assigned were... . each of these tubes were split into 3 tubes. With my ALW 148 tubes being split into 5 tubes. After the mini preps, also ran ligations. The samples that looked promising on the gel were sent for sequencing. After that was the step of PCR purification of my ALW148 sample.

we got additional dna from oliver that we needed to test out the concentration. The samples were...

7-2 Today we started our project of making fruiting bodies.

6-30 posted ‎‎Jun 30, 2009 8:29 PM‎‎ by Ethan Chan

Yesterday, we set up minipreps for our plates. When i came in this morning, i put all of my cloudy tubes into the centrifuge. When i took it out, there was a pellet at the bottem of each tube. We proceeded to do minipreps for our plates. I was in charge of plate 43-50. Everything went smoothly. After setting up the mini preps, we did a digest. The reaction was.... We created a master mix to save time. Ran it on a gel and printed out pictures. The ones that look sucessful will be sent to the lab for sequencing.

We also prepared for mini-preps for the 14 plates edna and i were assigned. We added 5ml media to each tube ( with correct antibotics) and then poked single colonies into each one. we did 3 tubes for each plate. for my alw148 plate, we did 5 tubes. These tubes will grow overnight so we can do the mini prep tomorrow.

6/29 posted ‎‎Jun 29, 2009 10:14 PM‎‎ by Ethan Chan

Continue to split the cells again. Currently at p6. We washed the plate with the old media serval times to get the cells loose, and then discarded the old media. We then added 12mls of new media. The Control plate only got media with G418. the other plates all had media with G418+hydro. Back in the fridge to grow overnight.

We were assigned plates with a partner. I am partners with edna. The main point of this lab was to get the entry vector into the Destination vector using the Gateway LR reaction. Many things were added into a tube ( see protocol) and then we proceded with a heat shock. After the heat shock, we used competent cells and allowed the reaction to recover. We added a few more things and then Incubated the tubes for about an hour. During that hour, we got ready for the plating, and added beads. Once the tubes were done incubating, we added 250ul from each tube to its corresponding plates. Those plates were placed in the incubator overnight to grow.

We prepared Mini-preps today for the vectors that didnt work. each of us had an assigned pHO number. mine was pHO50. We each took 10 tubes, added 5mls of media to each of them. and then placed a single colony to each of the tubes that will grow overnight.

6/19 to 6/26 posted ‎‎Jun 26, 2009 9:14 PM‎‎ by Ethan Chan

6/19 to 6/23 (in Vancouver)Some of the students sent me updates during my trip and noted that Alex did split the dicty cells because they were becoming too confluent.

6/24 We redid some of the digest because they still didnt show a clear result. Four of the samples didnt work, so we split them into 10 tubes each to hopefully get a better chance at suceeding. we had a total of 40 tubes and then set up a master mix.

Water 5.3ul 63.6ul Buffer 1ul 12ul PVU I 0.2ul 2.4ul DNA 3ul BSA: 0.5ul 6ul

6/25

I ran our 40 samples on a gel today. we ran the gel at 135v. The gel was premade at 2%, so inorder for us to make it back to 1%, we added 12.5 with buffer, and filled the rest up to the 25 mark with 2% agarose. While i did this, Alex proceeded to split our dicty cells once again. When the gel was done running, we took pictures of the gel.

6/26 Each of the Dicty team were assigned plates to be in charge of. We got new plates, added 11.5ml of Media with __418. we then took .5 mls of cells from the orginal plates and plated them in the new media.

What we did so far from 6/15 to 6/18 posted ‎‎Jun 18, 2009 3:34 PM‎‎ by Ethan Chan [ updated ‎‎Jun 26, 2009 9:12 PM‎‎ ]

6/16 Oliver taught us how to grow Dicty cells today. We outlined a colony from one of his plates and placed them in new media. My partner for this dicty project is Alex Smith. We placed them in the fridge.

In the lab, we prepared our mini preps. We added new media and then poked a single colony and placed them in indivdual tubes to grow overnight.

6/17 MINI-PREP: After growing the cells overnight, we took them out from the incubator early in the morning to put them in the fridge. We didnt want the cells to over grow and die so we prevented that by spinning them down, and then pouring off the supernantent. We put all the tubes back in the fridge. After the meeting, we prepared mini-preps. I really liked the vacume machinese that they have in the lab. It saves us time because we can elute the DNA quickly without centrifuging each of the tubes.

We checked our dicty cultures in the fridge later on in the day, and i was able to see dicty cells growing on the bottem of the plate. We were able to view them under the microscope, and they looked pretty healthy. They actually look a lot like the CHOK1 cells i was working with at city college. It seemed very similar.

6/18 Today we did our group digest. In the pre-projects, i was partnerd with eric. Since some of our results werent clear, we redid some of them. We digested purified plasmids that we got from the mini prep yesterday. To save time, we created a Master Mix

PVU I Digest: water 2.3ul buffer 1ul pvu 1 0.2ul DNA 3ul BSA .5ul

total: 10ul each tube