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Revision as of 19:50, 21 September 2012
Bacterial
- Placeholder1
- Placeholder2
in vitro
- P3
- P4
Mammalian
- Spring TC Notebook
- Summer TC Notebook
- Fall TC Notebook
HIHIHI
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.
Week Three: 6/25/12 - 7/1/12
June 25th, 2012
Made 5 cryovials of HEK293+EYFP.
June 27th, 2012
corrected media today. FBS was at 8.8%, so we created another bottle of media at 11.2%, then combined and filter sterilized together. Made FBS aliquots.
June 28th, 2012
Cells more confluent with new 10% media! Split six 1:6 plates for Saturday. Need a P1000 in the hood and need P20 calibrated
June 29th, 2012
Split two 1:6 plates of HEK293 for Sunday and two 1:6 plates of HEK293 eYFP for Sunday. Aliquoted more FBS. Will need more media soon ( we have another 350 mL bottle...) Got a new calibrated P20! Also aliquoted more trypsin.
June 30th, 2012
Split two 1:6 plates of HEK293 for Monday and two 1:6 plates of HEK293 eYFP for Monday. Also seeded a 6 well plate for FACs training. Should have 200,000 cells for each sample by Monday. Also did a media change on the ROX transfection, and took some images using the microscope in the TC room. Saved on the flashdrive there. Accidentally spilled bleach while cleaning the vaccum trap. Contained with paper towels, and cleaned up. (only spilled around 5-10mL). Also, our box of short pastuer pipettes is low, will need to autoclave more soon.
July 1st, 2012
Split two 1:6 plates of HEK293 for Tuesday and two 1:6 plates of HEK293 eYFP for Tuesday. Also seeded another 6 well plate for FACs training, but this time a higher density. Will probably pool samples for tomorrow. Should have ~1,000,000 cells for between the two dishes for tomorrow. Disposed of old (turning basic) media. Did FACS analysis and imaging of the ROX transfection that compares RNAiMax and Lipo2k.
Week Four: 7/2/12 - 7/8/12
July 2nd, 2012
Split two 1:6 plates of HEK293+eYFP for Wednesday. Split two 1:6 plates of HEK293 for Wednesday. Made a new 500 mL bottle of DMEM with phenol red with 10% FBS, 1% Nonessential Amino Acids, 1% L-Glutamine, 1% Pen/Strep
July 3rd, 2012
Split two 1:6 plates of HEK293 for Thursday. HEK293+EYFP were a bit under confluent. Also brought up a frozen stock of HEK293+EYFP to test cell viability.
July 4th, 2012
Split two 1:6 plates of HEK293 and HEK293+EYFP for friday.. HEK293+EYFP were a bit under confluent, let go till tomorrow. Did a media change on the transfections. Transfections in phenol red media were not as intensely red as before, only slightly tinted. Imaged and ran FACS analysis on transfections. Seeded a 6 well plate with ~ 500,000 cells per well for FACS training.
July 5th, 2012
Split two 1:6 plates of HEK293 and HEK293+EYFP for Saturday. Cells brought up from frozen stock on 7/3/12 about 70% confluent. Will also check again on 7/6/12. Ran FACS Analysis on Transfections from 7/3/12. Received Alexa tagged output for our Rep6. Diluted and annealed with the ROX tagged gate. Will transfect tomorrow. Also expecting LRs to transfect tomorrow.
Week Seven: 7/22/12 - 7/29/12
Sunday July 22nd:
Maintenance: We froze down 10 stocks of HEK293's at P3, which are in the -140. We have one plate of HEK293+eYFP split 1:6 today to be ready Tuesday. We have four plates of HEK293's split 1:6 for Tuesday. We have four plates of HEK293's split 1:3 for Monday. We have one plate of HEK293's split Friday ~1:6, which were under confluent, which should be ready tomorrow. Need to bring up one stock of 293's on Thursday to check for viability. Also need to remember to seed HEK293+eYFP's for Jonathan for the 26th. Also need to figure out the proper amount of L-Glutamine to add to the clear media. Prepared KB's Optimizing efficiency of dsRNA + DNA samples for FACS.
Monday July 23rd:
Split 4 plates of HEK293s 1:6 Ran FACS on KB's ALEXA, ROX, ALEXA:ROX, +S1, +S6 experiments (following preparation protocol from above)
Tuesday July 24th:
Took one plate of HEK293's at P4 (that were split 1:3) to freeze down into 10 cryovials, with ~1,000,000 cells each Transfections: KB repeated ALEXA/ROX transfections with 24 well plates (details on expt page) NK repeated the rTTA3 system, this time with a DOX curve Passaging: Split four plates of HEK293s at 1:6 for Thursday Split one plate of 293+eYFP for Thursday FACS Prep: Seeded 250,000 cells per well in a 6 well dish for Jon and whoever else wants FACS training on Thursday Seeded 2 wells of HEK293s, 2 wells of HEK293+eYFP, 2 wells of half 293s & half eYFPs. Will prep for FACS on Thursday
Wednesday July 25th:
1. Moved frozen down cells from -80 to -140 (at 9:00 A.M.) Note: Next Tuesday we should bring up a vial to test viability - KB - 7/26 2. Performed a media change on 9 wells of KB's Alexa, Tag transfections from 7/24 (at 10:30 A.M) 3. Supplemented Red Media (12:00 PM): 4. Supplemented Clear Media (12:00 PM): 5. Supplementing Media Notes: Keep all components in small aliquots - easy to thaw Always aliquot the entire FBS bottle - it is bad to keep thawing/freezing FBS 6. Eta transfected constitutive colors at 2:00 PM for Friday Microscopy Training 7. Katie transfected Alexa:ROX into her Tag well at 2:30 P.M. to optimize dsRNA + DNA transfection 8. Linh retransfected the eYFP knockdown system with FF1 to show that her efficiency and results are repeatable 9. Nathan induced a DOX ladder to his rtTA3/DOX system and also retransfected the entire system (due to volume issues the previous day) 10. Split 5 plates of HEK293's 1:6
Thursday July 26th:
7:40 - 8:40 A.M. - Prepare Katie's ALEXA FACS samples and Nathan's DOX FACS Samples 10:45 A.M. - 12:00 PM - FACS KB and NK samples 2:00 PM - NK: Prepare FACS samples for JE + AS We prepared 2 wells of 250k HEK293s. 2 wells of HEK293s+EYFP. 2 wells of mixed both 293 and 293+EYFP 4:30 PM - KB: Transfect S6 Input annealed to gate with Hef1a:eYFP Marker 4:45 PM - KB: Split four plates of HEK293's 1:6. Split one plate of HEK293+eYFP 1:6 5:00 PM - Nathan induced DOX ladder on his rtTA3 transfections
Friday July 27th:
8:00 AM - LV + NK prepare FACS samples 11 AM - FACS NK's rtTA3 DOX, LV's FF1 KD 2:00 PM - KB Transfect Alexa:ROX, +S1, +S6 system with 2X input. Transfect S6:Gate to well transfected with Hef1a:eYFP yesterday. • Protocol - Transfection72712.xlsx 3:30 PM - LV Transfect miRFF1 - eYFP knockdown system 4:30 PM - NK transfect three plasmid / four plasmid constitutive colors to up efficiency. Let's break 90%! Also retransfect DOX curve system? 6:00 PM - KB passaged four plates of HEK293's 1:6 (to be ready Sunday)
Saturday July 28th:
FACS KB Optimization / 2x Input Experiments FACS EA's Single Plasmid Constitutive Colors Expt. Induce DOX (NK) Transfect multi plasmid efficiency (NK) Passage
Sunday July 29th:
* NK DOX FACS * LV Knockdown FACS * EA transfect constitutive colors for scope / FACS training * Passage
Week Eight: 7/30/12 - 8/5/12
Monday July 30th:
* FACS NK multi plasmid efficiency
* LV transfect FF1 KD with different parameters
* KB transfect ALEXA/ROX system with 3X input. Also try dsRNA + ssRNA optimization as suggested by Eerik.
* Passage
Tuesday July 31st:
* Confocal KB's transfection
* NK transfect efficiency & multiple dox conditions
* LV FACS practice from EA's single color transfections
* Passage
* Make new clear DMEM!
Wednesday August 1st:
* Confocal KB's transfections
* KB transfect eYFP
* NK induce DOX ladder
* LV transfect KD with Hef1A:TetR
* make red supplemented DMEM
* FACS LV's KD with reduced parameters, KB's two ALEXA:ROX expts (one with 15 pmol rep6 and 45 pmol of either input) (one with 30 pmol rep6 and 75 pmol of either input)
Thursday August 2nd:
* KB transfect in 35 mm dishes at 7:45 AM
* KB confocal with DM at 10:30 AM
* NK FACS DOX Ladder, efficiency
* NK transfect FF4 system, FF1 system
* Make new clear supplemented DMEM (Note, we only have one stock bottle left!)
* Split 4 plates of HEK293 at 1:6
Friday August 3rd:
* NK transfect :
* KB transfect 15,20,25,30 ALEXA:ROX
* LV transfect FF4 system?
* Split 4 plates of HEK293 at 1:6
Saturday August 4th:
* KB FACS with DM
* NK FACS
Week Nine: 8/6/12 - 8/12/12
Monday 8/6/12:
NK transfect DOX curve with 16 data points NK transfect FF4 system with varying amounts of decoy / miRFF4, and the Lac system KB transfect FF1 system Split 4 plates of HEK293s
Tuesday 8/7/12:
KB transfect 15,20,25,30 pmol of annealed reporter along with 3 positive controls - Transfection of Rep6+Alexa (July 5th) NK redo DOX transfection (with 16 data points?) NK induce DOX, IPTG Split 4 plates of HEK293s at 1:6 with Jenna Bring Giulio into TC - bring up a new vial of eYFPs and start passaging
Wednesday 8/8/12:
FACS KB FF1 and NK FF4 at 9:00 A.M. Supplemented red DMEM with Giulio FACS NK DOX Ladder and KB Alexa:ROX Jenna and Giulio split five plates of HEK293+eYFPs at 1:6 Eta retransfect FF4 Nathan induce DOX on yesterday's transfection KB retransfect ALEXA:ROX KB retransfect FF1 system (with TetRKrab this time) KB split four plates of HEK293s at 1:6
Thursday 8/9/12:
main culture underconfluent - only performed media change Eta and Nathan retransfected FF4 system FACS NK DOX ladder made supplemented clear media
Friday 8/10/12:
FACS KB A:R + 3X Inputs FACS KB FF1 Knockdown with TetR, TetR-Krab KB transfect A:R + U6:TetO inputs KB transfect FF1 with TetR, TetR-Krab and DOX in media NK transfect LacI? FF4, 16 data point DOX GA transfect constitutive colors Passage - 5 plates 1:6 for Sunday
Saturday 8/11/12:
Scope KB A:R with DNA inputs FACS EA FF4 NK induce DOX ladder Design meeting! Passage - 5 plates 1:6 for Monday
Week Eleven: 8/20/12 - 8/26/12
Monday 8/20/12:
Split 2 plates of 293s at 1:6 Split 1 plate of eYFPs at 1:6 Giulio split 293s 1:6
Wednesday 8/22/12:
Split 1 plate of eYPFs 1:12 for Saturday Split 1 plate of 293s 1:3 for Thursday Split 2 plates of 293s 1:12 for Saturday Seeded a 6 well plate for Jameel. One well of 1,000,000 293s and one well of 1,000,000 eYFPs Giulio split 293s Things to do: make media (thaw FBS!) bring up cells from frozen check if there is lab stock of FBS
Thursday 8/23/12:
giulio microinjection expt
Saturday 8/25/12:
made supplemented clear DMEM, supplemented red DMEM Split one plate of 293s 1:3, two plates 1:6 and one plate 1:12 Split one plate of eYPFs at 1:12 Thawing a bottle of FBS to aliquot
Sunday 8/26/12:
made 50 mL aliquots of FBS for iGEM split cells (on P20) 1:3, 1:6 and 1:12 transfected constitutive colors for imaging
Week Twelve: 8/27/12 - 9/2/12
Tuesday 8/28/12:
Split cells (on P21) 1:3, 1:6 and 1:12
seeded cells for Giulio for FACS training on Thursday
Wednesday 8/29/12:
Giulio : microinjection experiment with HeLa cells
image the constitutive color transfection
transfect A:R in triplicate
image A:R (2 - 9 PM)
FACS at 5 PM
make media
bring cells up from frozen stock
plate HeLa cells
9/20/12
NK transfect FF1 titration expt
Split 3 plates of 293s at 1:6
9/21/12
NK transfect FF1 titration expt
Split 3 plates of 293s at 1:6
GA and KB nucleofection of long reporter, long reporter + bulge, with either the correct or incorrrect input
KB transfect U6-TetO-S1 and U6-TetO-S6
NK transfect FF1 titration expt
Split 3 plates of 293s at 1:6
9/21/12
NK transfect FF1 titration expt
Split 3 plates of 293s at 1:6
GA and KB nucleofection of long reporter, long reporter + bulge, with either the correct or incorrrect input
KB transfect U6-TetO-S1 and U6-TetO-S6
