"
Team:MIT/Notebook
From 2012.igem.org
Bacterial
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in vitro
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Mammalian
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April 9th, 2012
We are using HEK cells with constitutive EYFP. Typically, they need to be passaged 2-3 days, today we passaged at 1:6 ratio.
April 11th, 2012
Split at a 1:6 ratio. They will need splitting again by Friday. Also, iGEM now has P1000 tips and culture dishes in the bottom drawer. All of our goods are labeled "iGEM." The P1000 tips are to be used with the aspirator when aspirating liquids in the hood. Be sure to EtOH before and after use. Last night (4/11/12) Kenneth metafected cells at 11 pm. Movies are here: http://www.youtube.com/playlist?list=PL07C09A5037691E54
April 13th, 2012
Split at 1:12 ratio, so they will be able to last 3 days (till Monday). Made 10 Aliquots of Typsin (10 mL). They are on the bottom shelf of the freezer, labeled with iGEM on them. When you need to passage cells, take an aliquot and thaw in the hot water bath. For now, we won't be needing all 10 mL in the aliquot so I need to check if we should freeze it or put in the fridge.
April 16th, 2012.
Made media according to the formula above. Katie split cells today at 1:6. They need splitting on Wednesday. Also, materials for making media (except DMEM, and essential AA's), will be stored in aliquots in the freezer, labeled. Essential AA's will be aliquotted and stored in the +4. To make media, you will simply need remove the 50 mL of DMEM, and place that into a labeled conical tube. Then add thawed frozen components and essential AAs. Frozen components are on the bottom door shelf of the freezer. Essential Amino Acids are in the fridge on the door as well. DMEM can be found in the cold room, some is stocked in the metal min-fridge in the back too.
April 18th, 2012
Split cells at 1:6 ratio. Needs to be split again Friday. Also expanded what we have in culture. A back up dish will be maintained by myself only. Another back up dish will be maintained by Katie only. The plate labeled for experiments can have cells taken from it for transfections etc. When this is used, please notify me, so I can replace it.
April 20th, 2012.
Katie and I (Nathan) split cells today at a 1:13 ratio. They need to be split again on Monday. April 23rd, 2012. Nathan split his cells 1:6 (the petri dish labeled NK and the one labeled experiment) and will split again on Wednesday Katie split her cells 1:3 to prepare for transfection tomorrow Followed same protocol as above.
April 24th, 2012.
Katie used 2 mL of her cells that she did not use for transfection to split in a 1:6 ratio (2 mL cells, 11 mL of media) following standard protocol Katie also performed two transfections: (1) ROX+RNA(Gate) + CFP into her HEK293 cells (2) just CFP into her HEK cells with the help of Kenneth. Deepak is assisting with the confocal, the Zeiss and FACS.
April 25th, 2012.
Katie split the experimental plate 1:6 at 7:30 PM. Need to be split again on Friday. Nathan split a backup plate around 3 pm. Need to be split again on Friday.
April 26th, 2012.
Katie performed 5 transfections (using her culture). Katie also split her cells 1:3. They need splitting tomorrow as well as do Nathan K's and the experimental plates.
April 27th, 2012.
Nathan split a Back up and experimental plate were both split at 1:12 Katie split her plate 1:12.
April 30th, 2012.
Katie split her plate 1:6 in preparation for Ala's Transfections on Wednesday.
May 2nd, 2012.
Katie split her plate 1:3 in preparation for Ala's Transfections on Thursday.
May 3rd, 2012.
Katie performed 3 transfections of ROX with Ala'a. One with 0.1 ug ROX, one with 1 ug ROX and one with 3 ug ROX. Each also with 5 uL of CFP. Katie also split HEK cells (labeled KB) 1:3. Will split again on Friday
May 4th, 2012.
Nathan split cells 1:12. Will be ready by Monday. Will probably need to make another bottle of media by Monday. Katie split cells 1:12. Will be ready by Monday.
May 7th, 2012.
Nathan split cells 1:6. Will be ready by Wednesday. Also made a new bottle of media. Its labeled iGEM, Fully supplemented (with L-glu, Non-Ess. AA, FBS, P/S), and 5/7/12 Katie split cells 1:3. Will be ready tomorrow for transfection
May 8th, 2012.
Katie split cells 1:12. Katie also did 5 transfections (repeated from April 26th)
May 9th, 2012.
Nathan disposed of Katie's cells in culture. Split two plates 1:6.
May 11th, 2012.
Reducing what we have in culture to one plate. Split 1:12, will be ready on Monday.
May 14th, 2012.
Cells were quite confluent (95%), and split on the lower side of ~1:6 (2 mL from 13 mL total). Will be ready by Wednesday Also seeded a plate 1:3 for Katie, which will be ready by Tuesday to use in transfections. Also made more trypsin aliquots.
May 15th, 2012.
Katie used Nathan's cells that were seeded 1:3 for 5 transfections. All Details on [RNA & ROX Lifetime Experiment|igem2012:RNA & ROX Lifetime Experiment] page
May 16th, 2012.
Cells in dish marked NK were underconfluent. Possible that they were either split too hard, over trypsinized, or over confluency was no good. Media color is fine (Red), so will come back tomorrow and see if they need to be split or not. Also need to get them back onto a MWF splitting schedule. New aim is to re suspend in 12 mL total to make 1:6, 1:12 splits more straightforward. Plan is to split 1:3 Thursday, which means they would be confluent by Friday. Then split 1:12 for over the weekend. This of course depends on the confluency reached by tomorrow. Could also do a split that is 1:24 (which would last 4 days roughly before splitting), and see how that turns out by Monday.
May 17th, 2012.
Cells in NK dish were properly confluent (80%). Seeded two plates, one at 1:3, which will then be split tomorrow at 1:12. The other 1:24, and to be safe/relatively consistent with the other plate, will do a media change tomorrow. By Monday, going to check which looks more viable in terms of media colour, confluency etc. Then going to decide which to keep. (Serial splits, or one large split)
May 18th, 2012.
Cells in 1:3 were confluent... Pipetted up and down to detach from the plate instead of trypsin (don't want to loose healthy cells that may not have adhered). Final volume was 10 mL, so split 1:10. I checked under the scope before placing in the incubator, and they look similar to the 1:12 splits, so they should last until monday. To be safe, will check on Sunday their confluency. 1:24, changed media. But it is possible that some cells were washed away in the process (especially if they didn't adhere). Will check sunday as well, but the populations looked incredibly low. It may make sense for the summer to bring up a new stock from the freezer (depending on how high passage numbers are getting from my plate, which is all we have in culture now).
May 19th, 2012
Both cell populations looked incredibly low. The 1:24 split obviously has less cells than the serial split. However, neither were even 50% confluent, and the media still looks rather healthy. Will probably check on Monday for media color and see if a media change is necessary.
May 21st, 2012
Both cell populations looked low, but the serial split plate looked better than the 1:24. Changed media in the 1:24. Trypsinized the serial split to break up the large clumps of cells to spread more evenly in the plate, but did not split at a ratio (13 mL of cell solution went back into the dish). Will check on Wednesday.
May 23rd, 2012
Serial split plate was destroyed, none of the cells adhered/recovered from trypsinization. 1:24 plate has low populations, but looks OK. Did a media change, will check again on either Thursday afternoon or Friday.
May 25th, 2012
Media color is normal, but not changing at all. Cells are alive, but they are not spreading out on the plate. More or less forming "colonies." Today, they were trypsinized to break up the colonies, spun down at 260g (according to this Penn iGEM protocol ) for 5 minutes and then resuspended in fresh media. The entire solution was placed into the dish. Will check tomorrow afternoon their confluency. At this point, if these cant come back it would be wise to get a fresh stock of 293Ts. We also should make frozen DMSO stocks so that when mistakes occur, we can at least work from the same passage.
May 26th, 2012
Cells in culture look good! Growing more as a monolayer and not colonies. Looking about 70%-75% confluent. Will split 1:6 today (out of 12 ml) and check again on Monday. Hopefully we can get them back on the MWF schedule. Also as an extra precaution, cells were centrifuged out of the trypsin/media mixture and resuspended in 12 mL fresh media prior to seeding two tissue culture dishes.
May 28th, 2012
Cells look good. Both dishes were split 1:6 (out of 12 mL). Followed the normal protocol (no centrifuging). During the protocol, there was a problem with the pipette aid. It would aspirtate, but not dispel fluid. Had to grab a pipette aid from the hood next to the microscope (washed with EtOH when transferring to and from hoods). Emailed Patrick about it.
May 30th, 2012
Patrick emailed over info about what was going on with the pipettor in the hood. Cells are growing, but a bit underconfluent (50%-60%). Since I can not come into lab tomorrow, going to split 1:6 anyways, check on Friday. If still underconfluent a bit on Friday, will let go to Saturday. I would rather have the cells be underconfluent and in log phase than overconfluent and in a lag phase.
June 1st, 2012
One plate was allowed to grow for an extra day. The other was split 1:12, into two dishes to last until Monday (which they will be back at ~60% confluency). This gives us a backup plate in the event that letting a plate go an extra day means the cells are over confluent.
June 2nd, 2012
The plate that was allowed to go the extra day looks good, 80%-90% confluent, and not overgrown at all. Will Split 1:6 today, which will put back on MWF splitting schedule. Cells split yesterday will be checked on Monday... Could do either a 1:3 split from the backup plates on Mon. to get the 80-90% confluency by Wed. or simply expand our cultures from the one plate that looks good (80-90% confluent as of today). Expanded cultures from the properly confluent plate.
June 4th, 2012
Split cells 1:6. Will check on Wednesday.
June 6th, 2012
Made a new bottle of media. Split cells 1:6. Will check on Friday.
June 8th, 2012
Cells were underconfluent and allowed to grow an extra day.
June 9th, 2012
Cells are still underconfluent. Seeded a dish 1:3, the other 1:6. To ensure an accurate 12 mL final volume, cells in the trypsin/media mixture were centrifuged at 260g for 5 mins, and then resuspended in fresh media prior to seeding. Also, I dropped our aspirating tool on accident, and the glass portion to it broke. Put it in the pickle jar sharps container. Will need to make another one, or simply use pastuer pipettes. Also received an important email from Kenneth regarding the incubators. Information from said email is below. The water tray in the bottom of the incubator needs to be checked and filled when empty. Without water, the incubator will not be at the proper humidity and the CO2 sensor will not work properly, which means no CO2 and the media becomes too basic (purple) and cells will die/grow slowly. You can use the DI water from the white tap on the sink, no need to add anything to the water. Also, use a graduated cylinder to make easier! (need to check about antibacterial blue stuff)
We are using HEK cells with constitutive EYFP. Typically, they need to be passaged 2-3 days, today we passaged at 1:6 ratio.
April 11th, 2012
Split at a 1:6 ratio. They will need splitting again by Friday. Also, iGEM now has P1000 tips and culture dishes in the bottom drawer. All of our goods are labeled "iGEM." The P1000 tips are to be used with the aspirator when aspirating liquids in the hood. Be sure to EtOH before and after use. Last night (4/11/12) Kenneth metafected cells at 11 pm. Movies are here: http://www.youtube.com/playlist?list=PL07C09A5037691E54
April 13th, 2012
Split at 1:12 ratio, so they will be able to last 3 days (till Monday). Made 10 Aliquots of Typsin (10 mL). They are on the bottom shelf of the freezer, labeled with iGEM on them. When you need to passage cells, take an aliquot and thaw in the hot water bath. For now, we won't be needing all 10 mL in the aliquot so I need to check if we should freeze it or put in the fridge.
April 16th, 2012.
Made media according to the formula above. Katie split cells today at 1:6. They need splitting on Wednesday. Also, materials for making media (except DMEM, and essential AA's), will be stored in aliquots in the freezer, labeled. Essential AA's will be aliquotted and stored in the +4. To make media, you will simply need remove the 50 mL of DMEM, and place that into a labeled conical tube. Then add thawed frozen components and essential AAs. Frozen components are on the bottom door shelf of the freezer. Essential Amino Acids are in the fridge on the door as well. DMEM can be found in the cold room, some is stocked in the metal min-fridge in the back too.
April 18th, 2012
Split cells at 1:6 ratio. Needs to be split again Friday. Also expanded what we have in culture. A back up dish will be maintained by myself only. Another back up dish will be maintained by Katie only. The plate labeled for experiments can have cells taken from it for transfections etc. When this is used, please notify me, so I can replace it.
April 20th, 2012.
Katie and I (Nathan) split cells today at a 1:13 ratio. They need to be split again on Monday. April 23rd, 2012. Nathan split his cells 1:6 (the petri dish labeled NK and the one labeled experiment) and will split again on Wednesday Katie split her cells 1:3 to prepare for transfection tomorrow Followed same protocol as above.
April 24th, 2012.
Katie used 2 mL of her cells that she did not use for transfection to split in a 1:6 ratio (2 mL cells, 11 mL of media) following standard protocol Katie also performed two transfections: (1) ROX+RNA(Gate) + CFP into her HEK293 cells (2) just CFP into her HEK cells with the help of Kenneth. Deepak is assisting with the confocal, the Zeiss and FACS.
April 25th, 2012.
Katie split the experimental plate 1:6 at 7:30 PM. Need to be split again on Friday. Nathan split a backup plate around 3 pm. Need to be split again on Friday.
April 26th, 2012.
Katie performed 5 transfections (using her culture). Katie also split her cells 1:3. They need splitting tomorrow as well as do Nathan K's and the experimental plates.
April 27th, 2012.
Nathan split a Back up and experimental plate were both split at 1:12 Katie split her plate 1:12.
April 30th, 2012.
Katie split her plate 1:6 in preparation for Ala's Transfections on Wednesday.
May 2nd, 2012.
Katie split her plate 1:3 in preparation for Ala's Transfections on Thursday.
May 3rd, 2012.
Katie performed 3 transfections of ROX with Ala'a. One with 0.1 ug ROX, one with 1 ug ROX and one with 3 ug ROX. Each also with 5 uL of CFP. Katie also split HEK cells (labeled KB) 1:3. Will split again on Friday
May 4th, 2012.
Nathan split cells 1:12. Will be ready by Monday. Will probably need to make another bottle of media by Monday. Katie split cells 1:12. Will be ready by Monday.
May 7th, 2012.
Nathan split cells 1:6. Will be ready by Wednesday. Also made a new bottle of media. Its labeled iGEM, Fully supplemented (with L-glu, Non-Ess. AA, FBS, P/S), and 5/7/12 Katie split cells 1:3. Will be ready tomorrow for transfection
May 8th, 2012.
Katie split cells 1:12. Katie also did 5 transfections (repeated from April 26th)
May 9th, 2012.
Nathan disposed of Katie's cells in culture. Split two plates 1:6.
May 11th, 2012.
Reducing what we have in culture to one plate. Split 1:12, will be ready on Monday.
May 14th, 2012.
Cells were quite confluent (95%), and split on the lower side of ~1:6 (2 mL from 13 mL total). Will be ready by Wednesday Also seeded a plate 1:3 for Katie, which will be ready by Tuesday to use in transfections. Also made more trypsin aliquots.
May 15th, 2012.
Katie used Nathan's cells that were seeded 1:3 for 5 transfections. All Details on [RNA & ROX Lifetime Experiment|igem2012:RNA & ROX Lifetime Experiment] page
May 16th, 2012.
Cells in dish marked NK were underconfluent. Possible that they were either split too hard, over trypsinized, or over confluency was no good. Media color is fine (Red), so will come back tomorrow and see if they need to be split or not. Also need to get them back onto a MWF splitting schedule. New aim is to re suspend in 12 mL total to make 1:6, 1:12 splits more straightforward. Plan is to split 1:3 Thursday, which means they would be confluent by Friday. Then split 1:12 for over the weekend. This of course depends on the confluency reached by tomorrow. Could also do a split that is 1:24 (which would last 4 days roughly before splitting), and see how that turns out by Monday.
May 17th, 2012.
Cells in NK dish were properly confluent (80%). Seeded two plates, one at 1:3, which will then be split tomorrow at 1:12. The other 1:24, and to be safe/relatively consistent with the other plate, will do a media change tomorrow. By Monday, going to check which looks more viable in terms of media colour, confluency etc. Then going to decide which to keep. (Serial splits, or one large split)
May 18th, 2012.
Cells in 1:3 were confluent... Pipetted up and down to detach from the plate instead of trypsin (don't want to loose healthy cells that may not have adhered). Final volume was 10 mL, so split 1:10. I checked under the scope before placing in the incubator, and they look similar to the 1:12 splits, so they should last until monday. To be safe, will check on Sunday their confluency. 1:24, changed media. But it is possible that some cells were washed away in the process (especially if they didn't adhere). Will check sunday as well, but the populations looked incredibly low. It may make sense for the summer to bring up a new stock from the freezer (depending on how high passage numbers are getting from my plate, which is all we have in culture now).
May 19th, 2012
Both cell populations looked incredibly low. The 1:24 split obviously has less cells than the serial split. However, neither were even 50% confluent, and the media still looks rather healthy. Will probably check on Monday for media color and see if a media change is necessary.
May 21st, 2012
Both cell populations looked low, but the serial split plate looked better than the 1:24. Changed media in the 1:24. Trypsinized the serial split to break up the large clumps of cells to spread more evenly in the plate, but did not split at a ratio (13 mL of cell solution went back into the dish). Will check on Wednesday.
May 23rd, 2012
Serial split plate was destroyed, none of the cells adhered/recovered from trypsinization. 1:24 plate has low populations, but looks OK. Did a media change, will check again on either Thursday afternoon or Friday.
May 25th, 2012
Media color is normal, but not changing at all. Cells are alive, but they are not spreading out on the plate. More or less forming "colonies." Today, they were trypsinized to break up the colonies, spun down at 260g (according to this Penn iGEM protocol ) for 5 minutes and then resuspended in fresh media. The entire solution was placed into the dish. Will check tomorrow afternoon their confluency. At this point, if these cant come back it would be wise to get a fresh stock of 293Ts. We also should make frozen DMSO stocks so that when mistakes occur, we can at least work from the same passage.
May 26th, 2012
Cells in culture look good! Growing more as a monolayer and not colonies. Looking about 70%-75% confluent. Will split 1:6 today (out of 12 ml) and check again on Monday. Hopefully we can get them back on the MWF schedule. Also as an extra precaution, cells were centrifuged out of the trypsin/media mixture and resuspended in 12 mL fresh media prior to seeding two tissue culture dishes.
May 28th, 2012
Cells look good. Both dishes were split 1:6 (out of 12 mL). Followed the normal protocol (no centrifuging). During the protocol, there was a problem with the pipette aid. It would aspirtate, but not dispel fluid. Had to grab a pipette aid from the hood next to the microscope (washed with EtOH when transferring to and from hoods). Emailed Patrick about it.
May 30th, 2012
Patrick emailed over info about what was going on with the pipettor in the hood. Cells are growing, but a bit underconfluent (50%-60%). Since I can not come into lab tomorrow, going to split 1:6 anyways, check on Friday. If still underconfluent a bit on Friday, will let go to Saturday. I would rather have the cells be underconfluent and in log phase than overconfluent and in a lag phase.
June 1st, 2012
One plate was allowed to grow for an extra day. The other was split 1:12, into two dishes to last until Monday (which they will be back at ~60% confluency). This gives us a backup plate in the event that letting a plate go an extra day means the cells are over confluent.
June 2nd, 2012
The plate that was allowed to go the extra day looks good, 80%-90% confluent, and not overgrown at all. Will Split 1:6 today, which will put back on MWF splitting schedule. Cells split yesterday will be checked on Monday... Could do either a 1:3 split from the backup plates on Mon. to get the 80-90% confluency by Wed. or simply expand our cultures from the one plate that looks good (80-90% confluent as of today). Expanded cultures from the properly confluent plate.
June 4th, 2012
Split cells 1:6. Will check on Wednesday.
June 6th, 2012
Made a new bottle of media. Split cells 1:6. Will check on Friday.
June 8th, 2012
Cells were underconfluent and allowed to grow an extra day.
June 9th, 2012
Cells are still underconfluent. Seeded a dish 1:3, the other 1:6. To ensure an accurate 12 mL final volume, cells in the trypsin/media mixture were centrifuged at 260g for 5 mins, and then resuspended in fresh media prior to seeding. Also, I dropped our aspirating tool on accident, and the glass portion to it broke. Put it in the pickle jar sharps container. Will need to make another one, or simply use pastuer pipettes. Also received an important email from Kenneth regarding the incubators. Information from said email is below. The water tray in the bottom of the incubator needs to be checked and filled when empty. Without water, the incubator will not be at the proper humidity and the CO2 sensor will not work properly, which means no CO2 and the media becomes too basic (purple) and cells will die/grow slowly. You can use the DI water from the white tap on the sink, no need to add anything to the water. Also, use a graduated cylinder to make easier! (need to check about antibacterial blue stuff)
Week One: 6/11/12 - 6/17/12
June 11th, 2012
One plate of cells was underconfluent, will be ready by tomorrow (the 1:6 from 6/9/12). The 1:3 plate was 90% confluent. Seeded two plates at 1:3, two plates at 1:6, to have cells ready by tomorrow and Wednesday. The plan is to potentially use one plate tomorrow to seed more plates for Thursday and Friday, and expand what we have in culture. We need to have a conversation with Deepak to have a concrete plan on how much we need to expand our cultures.
June 12th, 2012
To Do: 1cm plate for Deepak/ other students both 1:6 plates appeared way under confluent on the scope (about 10% confluence) used 1:3 plate from 6/11/12 for transfections of pEXPR Hef1a:Cerulean and IGEM-BBa_K779100 (the gate i.e. the fluorophore + RNA)
June 13th, 2012
Cells are still underconfluent, and have reached a pretty high passage number (at least 30). So, we are talking about getting fresh cells from the summer, and this time making a DMSO stock so we can return to a low passage. We performed 6 transfections in duplicate using both 1:6 plates.
June 14th, 2012
Bleached and threw out all remaining 1:3 and 1:6 plates which we were maintaining Received a frozen DMSO stock of HEK293 (without constitutive YFP) from Kenneth (1.5x 10^6 cells). Going to make more DMSO stocks on Monday.: We thawed out the cell. Added them to 12 mL of DMEM with phenol red. Spun down at 1180 RPM for 4 minutes. Aspirated off the media / DMSO. Resuspended the cells in 12 mL of fresh DMEM. Transferred all 12 mL of culture onto a new dish. We also observed the Zeiss data from the transfections.
June 16th, 2012
As of today, we don’t have HEK+EYFP, but we may not need them Split HEK293 today 1:6 for monday. Seeded 5 plates, so that we have enough for transfections and to create DMSO stocks. Also added DIH20 to the tray at the bottom of the incubator, to keep humidity and CO2 levels constant. Also created more FBS stocks. One for Freezing Media, another for making media for normal culture.
Week Two: 6/18/12 - 6/24/12
June 18th, 2012
We split one plate of cells and seeded two dishes 1:6. One is back up, the other to potentially use for transfections on Wednesday. We also seeded a dish 1:3 to use in transfections tomorrow for imaging. We used three plates to make cell stocks of HEK 293T. 3 cryovials contain 1.2x10^6 cells. 3 cryovials contain 1.5x10^6 cells. One plate was then used for transfections for FACs. A static image was also taken.
June 19th, 2012
We seeded one plate 1:6 to be ready by Thursday. We also repeated the transfections for FACs analysis and Continuous imaging.
June 20th, 2012
Prepared samples for FACs analysis. We also split one plate 1:6 to have two dishes ready by friday for transfections. Today we also transfected eCFP to get a quantitative measurement of our transfection efficiency on Friday.
June 21th, 2012
Split 1:6 plate from 6/19 1:6 to be ready for Saturday.
June 22nd, 2012
split 1:6 two plates for Sunday (possibly Monday)
June 24th, 2012
There were HEK293+EYFP in our incubator, dated 6/22. As a result, the cells were very confluent, almost 100%. Cells were seeded at 1:3, 1:6 and 1:12. Hopefully, they are still viable. The 1:3 plate, if totally confluent again tomorrow can serve for cells tocks, the rest for transfections if necessary through the week. Or, making more cell stocks... Today I am also bringing up one of the cryovials of HEK293 to test cell viability. Another bottle of fresh media was made. The concentrations are slightly off, by around 1.25%, but according to Deepak it should be OK. Also autoclaved more pastuer pipettes.
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