20.109(F07): TA's notes for module 2

Expression Engineering Module

Current: 20.109(F07)
no wiki archive version, .docs from Spring 2005

General notes

Before term begins:

1. Check that all links on all wiki pages for expression engineering modulre properly direct to current version of class and to working webpages. Fix broken ones.
2. Order siRNAs in advance of module. These can take a while. 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/ul as follows: for each siRNA mix 40 ul of 5x siRNA buffer from Dharmacon with 160 ul 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 ul/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/ul. Students will need 1 ul 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 ul EB then pool. Measure concentration of 2.5 ul in 500 ul H2O (1:200 dilution). done for F'07: A260 of 1:200 was 0.02, giving stock conc of 0.2 ug/ul = 200 ng/ul 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/ul. Students will need 1 ul of working conc / transfected well in 6 well dish.
4. LAR/Stop and Glo: check -20 for aliquots and if needed order more.
5. MES cells and media, see notes for Day 1 and recipes
6. 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 enotugh 25 cm2 flasks/6 well dishes. Check that necessary solutions are there and are not expired: PBS, DMEM, trypsin, OptiMEM, gelatin, LIF, lipofectamine.
7. Qiagen prep for RNA
8. Qiashredders for RNA
9. 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.
10. Confirm there are some saved luciferase lysates for students to pre-run assays.
11. 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.
12. Confirm the microarray slides have been ordered.
13. Confirm the scanner is available for needed days.
14. Confirm Genisphere kit has been ordered.

Daily Notes

Notes:Day 1

Before lab:

1. need siRNAs ordered, have copy of spec sheets available so students can compare their design to what was ordered.
2. in preparation for lab on Day 2 you will also be seeding some 6 well dishes with J1 cells, grown in the absence of antibiotics (pre-txn media)...so be sure to look ahead to day 2 prep as well.

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 facililty:
   • 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.
   • aliquot PBS, trypsin, gelatin, J1 growth media.

Notes:Day 2

Before lab:

1. Need to aliquot luciferase assay reagents.
2. Need to aliquot lipofectamine, and OptiMEM and any cell culture growth reagents.
3. Need to aliquot psiCHECK plasmid and siRNAs. Each group needs minimum of 8 ul of diluted plasmid and 2 ul of diluted validated siRNA, and 2 ul of diluted scrambled siRNA and 4 ul of experimental siRNA. These are the minimal volumes.
4. 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.

Day of lab:

1. Need quiz (all quizzes 2 or 3 questions, 5 points).
2. Prewarm cell culture reagents in water bath or thaw DNA/RNA just before students will use them.Aliquot lipofectamine and OptiMEM as well.
3. Set up luminometers and luciferase assay reagents at RT.
4. Thaw lysates to be used in luciferase assays and leave in ice bucket near luminometer. Aliquot so each student group has three samples: a -/- (i.e. no ff, no renilla), a +/+ (both ff and renilla), and a +/- (renilla knocked down).
5. Luciferase Assay Reagents: 20ul into 980 ul buffer (already aliquoted and frozen in this volume).

Day after lab:

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

Notes:Day 3

Before lab:

1. Set out luminometer if put away.
2. Check there is sufficient bench paper.
3. 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.
4. Aliquot sterile water into 15 ml falcon tubes since the students can use this rather than RNase free water to measure RNA concentration.
5. 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 /student. Aliquot so each pair has amount they need, i.e. don't have students using common stock.
6. Just before lab: prepare RLT with BME, each pair of students will need 1 ml aliquot.
7. Just before lab: thaw luciferase reagents.

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).

Notes:Day 4

No prep except to read article for discussion. No quiz.

Notes:Day 7

No prep. No quiz.

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 ul 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 ul 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