TA's Notes for Module 2

General notes

Before term begins:

  1. Pre-run the design instructions for day one on all six sections of luciferase and confirm that the students will pick the siRNAs that we plan to order. It's SO important to know if the siRNAs are different, since these are the ones the students must follow all the way through the module. Analysis at the end of the module is much harder if the student's designs aren't the ones they actually use.
  2. Order siRNAs in advance of module. These can take a while. You can cut and paste the sequences from the Students iRNA file (PDF). Also the sequences are found in the following publication. When they arrive, use gloves to handle in RNase free way, spin briefly in microfuge to bring contents to bottom of tube then freeze as dry pellets. Dilute to 0.1 nmoles/µl as follows: for each siRNA mix 40 µl of 5x siRNA buffer from Dharmacon with 160 µl RNAase free water (these are stored at RT in RNA drawer), then add this volume to tube with siRNA. Vortex briefly then aliquot into RNase-free tubes (20 µl/tube), numbered "1 of 10" "2 of 10" etc. Store at -20 with rest of materials for module. Day of lab, dilute 1:10 in OptiMEM in hood in sterile tubes for working concentration of 10 pmoles/µl. Students will need 1 µl of working conc / transfected well in 6 well dish.
  3. Streak out NB165 on LB+Amp. This strain has psiCHECK2 dual luciferase plasmid. Make 4 overnight cultures (each 2.5 ml LB+amp). Miniprep these for the plasmid DNA using Qiagen DNA miniprep kit. Elute each with 50 µl EB then pool. Measure concentration of 2.5 µl in 500 µl H2O (1:200 dilution). done for F'07: A260 of 1:200 was 0.02, giving stock conc of 0.2 µg/µl = 200 ng/µl using 1 OD = 50 ug/ml. Day of lab, dilute 1:10 in OptiMEM in hood in sterile tubes for working concentration of 20 ng/µl. Students will need 1 µl of working conc / transfected well in 6 well dish.
    LAR/Stop and Glo: check -20 for aliquots and if needed order more.
  4. MES cells and media, see notes for Day 1 and recipes
  5. Prep TC room: this means make sure the 70% EtOH flasks are full, the pipets are stocked at each station, the traps are empty except for somme bleach in the flasks, autoclave more tips/tubes/pasteurs as needed, confirm there are enough 25 cm2 flasks/6 well dishes. Check that necessary solutions are there and are not expired: PBS, DMEM, trypsin, OptiMEM, gelatin, LIF, lipofectamine.
  6. Qiagen prep for RNA
  7. Qiashredders for RNA
  8. After last day of cell culture need to freeze away cells for next term. Grow 2 x 6 well dishes to ~ 80% confluence. Wash each well with 2 ml PBS. Trypsinize with 1 ml trypsin for 1' in hood then aspirate and incubate in incubator 10'. Triterate each well with 1 ml Freezing Media (J1 growth media + 20% serum + 20% TC grade DMSO found in cell culture room)(9 ml JI, 3 ml serum, 3 ml DMSO). Pool 2 ml into each cryotube and wrap in paper towels, pack into styrofoam case and freeze middle shelf of -80. Always want to freeze slowly and thaw fast. If there are samples of J1 cells in -80 that are >1 year old, destroy them.
  9. Confirm there are some saved luciferase lysates for students to pre-run assays.
  10. Thaw J1s by placing mixing 2 ml cryotube stock into 8 ml of J1 media in 25 cm2 flask previously treated with gelatin. Do this for 4 of 6 frozen stock tubes. Grow ON. Change media to dilute DMSO.
  11. Confirm the microarray slides have been ordered.
  12. Confirm the scanner is available for needed days.
  13. Confirm Genisphere kit has been ordered.

Daily Notes

Day 1

Before lab:

Need siRNAs ordered, have copy of spec sheets available so students can compare their design to what was ordered.

think through need for high G and low delta G in target sequence. Want area targeted to be naturally unfolded rather than all scrunched up. Since taking folded structure and unfolding it would take energy, presume unfolded to folding would release energy and so unfolded would have low G value and folded would have high G value. Going from unfolded to folded would have negative delta G.

Day of lab:

  1. No quiz on day 1
  2. In main teaching lab:
    • no prep, just computers with printer paper
  3. In cell culture facility:
    • each student will need 25 cm2 flask of ~ confluent J1 cells. So for lab of 12 students, prep 15 flasks so there can be 2 demo flasks as well and a back up flask in case of mistakes.
    • place hemacytometers and 20 µl pipet and box of tips by microscopes.
    • aliquot PBS, trypsin, gelatin, J1 growth media.
    • for Fall 2007 aliquots were:
      • 14 ml gelatin in 15 ml falcon, Marked "G"
      • 5ml trypsin in 15 ml falcon, Marked "T"
      • 12ml PBS in 15 ml falcon, Marked "P"
      • 15 ml growth media in 15 ml falcon, Marked "M." This was to stop trypsinisation.
      • 10 ml growth media in 15 ml falcon, Marked "M2." This was to be used for dilution of cells that the students would plate in 6 well dishes.

For each day of lab made 8 tubes like these, since only 6 students in TC at a time and the second crew used up the materials that the first group started. Other sets for teacher (needs only 1 demo set) and want at least one extra. Only exception: M2 tubes...need 12 of these/12 students since each student makes one dilution.

  • Preparation of J1 cells
    • Cells for both days (Tuesday, Wednesday for e.g.) can both be plated on Monday, you just have to make sure that the cell density for the Wednesday's slot is lower when you seed the cells. 3 flasks of confluent T25 J1 cells should provide you enough cells for both days.
    • For each confluenct T25 J1 cells, after trypsinisation, I resuspended them in 12ml of medium (this is my stock for Tuesday flask), take 1 ml of the stock and put it in a new flask containing 6ml of fresh medium (this is for the Tuesday student, you need to prepare at least X+2 flasks for a group of X student and for demonstration. Similarly, for the Wednesday group, I resuspended 1 confluent flask with 22ml of medium(this is my stock for the Wednesday's flask, note that it's more diluted than the Tuesday's stock), add 1 ml of stock to a new flask containing 6ml of fresh medium as described above.
    • Note that the above described is a very BIG split, but J1 cells grow really very fast!

Day 2

Before lab:

  1. Need quiz for day of lab.
  2. Each pair will need 2 six-well dishes of ~50% confluent J1 cells, growing in pre-txn media. So for lab of 12 students thats 12 dishes plus one or two in case of mistakes.
    • The most accurate rate to ensure that you have ~50% confluent J1 cells on the day of experiment is to try to grow some cells in advance to get a feel of how fast they grow and do the actual cell counting. For 20.109(F07), I resuspended one flask of fully confluent cell in 22ml of pre-transfection medium (this is my stock), take 1.5ml of stock and top up to 36ml with per-transfection medium. This 36ml is enough for me to plate two 6-well plate (3ml*6*2=36ml). I did this 2 days in advance (did on Sunday for Tuesday class). Cells for Wed class were also prepared on Sunday, I took 0.7ml of stock and top it up to 36ml with pre-transfection medium.
  3. Need to aliquot luciferase assay reagents (do this day of lab).
    • try 300 µl LARII/group and 300 µl Stop and Glo/group (20 µl/980 µl aliquots of buffer that are frozen -20).
    • try 50 µl of each sample/group. There should be 3 samples (1= no ff, no ren, 2 = high ff, high ren, 3 = high ff, lower ren)
    • all should be kept on ice until just before students try assays then move to RT
  4. Need to aliquot lipofectamine (50 µl/eppendorf, 12 eppendorfs/12 groups), and OptiMEM (1.4 ml/eppendorf) and any cell culture growth reagents
    • 30 ml PBS/falcon tube, 12 groups worth for both days of lab.
    • 14 ml Pre-Txn media/falcon tube, 12 groups worth for both days of lab.
  5. Need to aliquot psiCHECK plasmid and siRNAs (do this day of lab). Each group needs minimum of 8 µl of diluted plasmid and 4 µl of diluted validated siRNA, and 2 µl of diluted scrambled siRNA and 2 µl of experimental siRNA. These are the minimal volumes.
    • Plasmid dilution information:
    • stock plasmid (info is also above in "before the module starts" section): Streak out NB165 on LB+Amp. This strain has psiCHECK2 dual luciferase plasmid. Make 4 overnight cultures (each 2.5 ml LB+amp). Miniprep these for the plasmid DNA using Qiagen DNA miniprep kit. Elute each with 50 µl EB then pool. Measure concentration of 2.5 µl in 500 µl H2O (1:200 dilution). done for F'07: A260 of 1:200 was 0.02, giving stock conc of 0.2 ug/µl = 200 ng/µl using 1 OD = 50 ug/ml.
    • dilutions of plasmid: day of lab, dilute 1:10 in OptiMEM in hood in sterile tubes for working concentration of 20 ng/µl. Students will need 1 µl of working conc / transfected well in 6 well dish. Aliquots of plasmid should have ~10 µl each so can add 90 µl of Optimem to two just before lab and aliquot ~ 25 µl/epp/group.
    • siRNA dilution information:
    • stock siRNAs: These were ordered in 20 nmole quantities then diluted to 0.1 nmoles/µl as follows: for each siRNA mix 40 µl of 5x siRNA buffer from Dharmacon with 160 µl RNAase free water (these are stored at RT in RNA drawer), then add this volume to tube with siRNA. Vortex briefly then aliquot into RNase-free tubes (20 µl/tube), numbered "1 of 10" "2 of 10" etc. Stored at -20 with rest of materials for module.
    • each group will need one dilution of validated, one dilution of scrambled, and one dilution of student siRNA.
    • dilutions for siRNAs on day of lab, dilute 1:10 in OptiMEM in hood in sterile tubes for working concentration of 10 pmoles/µl. Students will need 1 µl of working conc / transfected well in 6 well dish.
      • student siRNAs 1-6: dilute one of each for each day of lab by adding 180 µl Optimem just before lab.
      • validated and scrambled siRNAs: dilute two of each for each day of lab by adding 180 µl of Optimem just before lab and then aliquoting 40 µl/eppendorf into 6 eppendorf tubes.

Day after lab:

Change media on student's tranfection plates to 3 ml J1 growth media.

Day 3

  1. Before lab:
  2. Set out luminometer if put away.
  3. Check there is sufficient bench paper.
  4. Put 3 open bottles of RNAse away at the front bench.
  5. Set up camera at dissecting microscope and set out card reader at front bench.
  6. Aliquot 30 ml of room temperature PBS in 50 ml falcon tubes/group. Leave in the teaching lab for the students to use when they wash the cells there before lysis.
  7. Aliquot sterile water into 15 ml falcon tubes since the students can use this rather than RNase free water to measure RNA concentration.
  8. Just before lab: 5X PLB to 1X with sterile water in 15 ml falcon tubes. Use a new bottle of water, sterile pipets for measuring the PLB and wear gloves to aliquot to minimize RNase contamination. Need minimum of 6 ml /pair of students. Make 10 ml/pair so there is plenty (2 ml 5X PLB + 8 ml H2O) Aliquot so each pair has 10 ml they need, i.e. don't have students using common stock.
  9. Just before lab: prepare RLT with BME, each pair of students will need 1 ml aliquot (= 10 µl BME + 990 µl RLT). Make this in the hood wearing gloves and in new, never touched eppendorf tubes with an RNAse-away cleaned pipetmen or the RNA only pipetment.
  10. Just before lab: thaw luciferase reagents. Each student pair should get 800 µl LAR and 800 µl S+G. See notes of Day 2 for how to prepare S+G. The LAR, if frozen, should already be good to go.

Day of lab:

  1. Need quiz
  2. Set out RNeasy kit and quishredders. Do not include any reagents that are not needed or are incomplete (e.g. still need EtOH added to them) since students have used these accidentally in the past.
  3. Midway through lab retrieve quartz cuvettes and turn on UV lamp on spec.
  4. Don't forget to turn UV lamp of spec off before leaving lab for the day.
  5. Freeze RNA samples at -20° in RNase-free box for next time. Should also freeze at -20° some of students luciferase lysates to use as samples next time module is run (label tubes 1 = -/- or 2 = +/+ or 3 = +/-)

Day after lab:

  1. Freeze some J1 cells for next time (see general notes above for details).

Day 4

No prep except to read article for discussion. Can offer a quiz either this day OR next but probably not both.

Day 5

  1. Microarray work.
  2. In advance of lab can autoclave 1X1L ddH2O in 2L flask and 2X2L in 4L flask.
  3. To sterile 1L of H2O, add 1 pkg of 20XSSC mix from Ambion (cat # 9764)
  4. Prepare 3L of 6X SSC +0.005% Triton X-100 (300 ml 20X + 50 µl T + 700 ml H2O)
  5. Prepare 2L of 2X SSC +0.0016%T (1:3 dilution of 6x)
  6. Prepare 2L of 0.2X SSC + 0.00016%T (1:30 dilution of 6X)
  7. Just before lab thaw RNAse free water and vials 11 (capture sequences), set one heat block to 80 and other to 42.
  8. At start of lab thaw vial 4 ("Superase") and materials for cDNA synthesis cocktail. For 15 reactions worth mix on ice:
    • 60 µl 5X buffer (comes with enzyme)
    • 15 µl dNTPs
    • 30 µl 0.1M DTT
    • 15 µl RT (leave in freezer until just before needed)
  9. Make fresh 0.5 M NaOH/0.5M EDTA (0.1g NaOH into 5 ml 0.5M EDTA). Aliquot 2x 100 µl.
  10. Aliquot 2x 100 µl Tris, pH7
  11. Aliquot 2x 200 µl Agilent 2X hyb buffer

Next day hyb solution

  • Prewarm 2X SDS hyb solution (vial 6) to 65° and fluors (vials 1) at RT before washing slide in BMC.
  • Fluor hyb, per array (x8)
    • 50 µl 2x hyb, vial 6 (400 µl)
    • 1.25 µl red vial 1 (10 µl)
    • 1.25 µl blue vial 1 (10 µl)
    • 48 µl H2O (384 µl)
  • Heat to 80° and at that time dry slide.

Day 6

No prep except to read lab in advance to get ready for data analysis.

Day 7

No lab. No prep.

Day 8

No lab. No prep.

Recipes/Reagents

  1. JI Growth Media: 500 ml DMEM (high glucose), 50 ml FBS (Atlanta Biologic, Inc.), 5 ml P/S/G, 1 ml BME, 5 ml NEAA. Filter then add 50 µl LIF. Store 4°C
  2. Pre-Transformation Media: 500 ml DMEM (high glucose), 50 ml FBS (Atlanta Biologic, Inc.), 5 ml 100XG, 1 ml BME, 5 ml NEAA. Filter then add 50 µl LIF. Store 4°C
  3. 0.1% gelatin for TC dishes > 10 min before adding cells: 1 g/L, Heat slightly to dissolve. Autoclave. Store 4°C