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

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# Change media on student's tranfection plates to 2 ml J1 growth media.
# Change media on student's tranfection plates to 2 ml J1 growth media.


===[[20.109(F07):_DNA_ligation_and_bacterial_transformation | Notes:Day 3]]===
===[[20.109(F07): Luciferase assays and RNA prep | Notes:Day 3]]===
Before lab:  
Before lab:  
# Have LB-Kan plates poured. Need 5/group = 60, 1L makes 30-40 plates, so need to make a couple liters.
#  
# Refill burners and jars with 100% ethanol
 
Day of lab:
# Need quiz
# '''Keep cold'''
#* 70% ethanol, 6 x 15 ml falcon tubes
#* 100% ethanol, 6 x 15 ml falcon tubes
#* From -20°C freezer:
#**students' inserts and backbone
#**T4 DNA ligase, 3 aliquots in PCR tubes in cold-boxes
#**T4 ligase buffer, has ATP so must be kept cold
#**BamHI, 2 aliquots in PCR tubes in cold-boxes
#**NEB Buffer 2
#**aliquots of yeast tRNA from 109 Antibodies box
#*'''only take out of -80°C freezer when first group is ready''', competency decreases with time on ice
#**super competent XL1-blue cells, one 200 ul aliquot per group
# Sodium acetate 3M, 6 x 100 ul in microtubes
# LB media, 10 ml per group, put in 50 ml conical tubes (3 x 30)
# Sterile water, 6 x 1 ml in microtubes for ligation and resuspending DNA
# LB-Kan plates out of 4C
# Kanamycin, 10 ul per group (2 tubes of 50 ul), '''keep cold'''
# large test tubes (4/group), 5 and 10 ml pipettes
# ice buckets for competent cells
# After incubating xformation plates for one day, blow colony plugs into overnights for use on Day 4. Students will have labelled tubes.
 
===[[20.109(F07):_Examine_candidate_clones | Notes:Day 4]]===
Before lab:
#Make 2L 1X TAE for gels and running buffer
#Pour 3 agarose gels, 1% in TAE (100ml) + 2 ul EtBr each. Use '''2 10-tooth combs'''/gel.
#Aliquot solutions (see below)
 
Day of lab:
Day of lab:
# Need quiz
# Need quiz
# Aliquot solutions for miniprep (Solutions 1, 3, NaOH and SDS) for each group so stocks don't get contaminated.
#  
#*Each group needs:
===[[20.109(F07): Journal article discussion | Notes:Day 4]]===
#**400 ul Solution 1 (6 microtubes of 1000 ul)
No prep except to read article for discussion. No quiz.
#**500 ul SDS 2% (6 microtubes of 1000 ul)
#**500 ul 0.4M NaOH (6 microtubes of 1000 ul)
#**600 ul Sol 3 (6 microtubes of 1000 ul)
#**4 ml 100% Ethanol (6 x full 15 ml tubes)
#**2 ml 70% Ethanol (6 x ~10 ml in 15 ml tubes)
#**sterile water bottle for each group
#Keep cold:
#*buffers
#*restriction enzymes
# ice buckets (one per bench should be fine, just for miniprep)
# 1 ml serological pipets for alcohols (optional)
# loading dye for gels
 
===[[20.109(F07): M13.1 | Notes:Day 5]]===


===[[20.109(F07):_Western_analysis | Notes:Day 6]]===
===[[20.109(F07): cDNA synthesis and microarray| Notes: Day5]]===
Day before lab:
# Start overnights of 7x NB271 liquid cultures for plaque assay (3 ml of LB+Kan in large tubes), each group needs ~2 ml cells.
# Start overnights of 7x NB251 liquid cultures for + control on Western (3 ml of LB+Kan in large tubes), each group needs ~1 ml.
# Pour 2L of LB plates
# Have protein gels and chambers
# For each day, make microtubes of 1X sample buffer without BME (500 ul 2X + 400 ul H20) add 100ul of BME to both on day of lab (could parafilm after adding BME, save 2 days). Need <1 ml / group.
# Make 3 L of transfer buffer for each day and refrigerate
# Make 1L 1X TBS-T in graduated cylinder, refrigerate, mix in 5% powdered milk close to lab day


* Note: 2°Ab is goat antimouse (GAM-AP) in ab freezer box
===[[20.109(F07): Microarray data analysis | Notes:Day 6]]===


Day of lab:
===[[20.109(F07): Mod 2 Day 7| Notes:Day 7]]===
* Need quiz
No prep. No quiz.
* Turn on water baths and melt top agar
* Protein gels and blot
# Students' candidates in liquid culture tubes
# Blank for spec (900 ul H2O and 100 ul LB)
# Sample buffer + 100 ul BME
# Lid locks for microtubes in hood
# Boiling tank on hot plate in hood with boiling chips, be sure it's turned off once students are done.
# 2 protein gel chambers with 2 gels in one and 1 plus spacer in other, set up
# "Kaleidoscope" protein molecular weight standard, in -20C in enzyme rack, aliquot 2x50 ul in microtubes, denature by boiling when students boil their samples
# Running buffer, 1X TGS made when needed, 1L/chamber, 10X above sink
# Transfer cassettes, ScotchBrite pads, filter paper, nitrocellulose
# Western transfer buffer, 1L/tank so 3L/day
# ice packs for Western
# Blocking buffer TBS-T + 5% milk, 50 ml/group
# Protein gels will have two groups/gel so that blot can be cut in half, and each probed with anti-p3.
* Plaque assay
# top agar, melted in microwave and kept in 55C water bath
# 200 ul x 10 /group NB271(=ER2267) bacteria for plaque assay
# 10^6 dilution of positive phage control in microtube
# small sterile test tubes, 10/group
# 10 LB plates/group for plaque assay (no phage, + phage control, 4 dilutions supernatant: 10^0,2,4,6, for each of two candidates)
# 5 ml pipets and Pipetmen
 
===[[20.109(F07):_Probe_western | Notes:Day 7]]===
 
* EHS rep will come in and talk about cell culture work during first Western incubation period.
* Talk about M13 refactoring during other incubation
 
Day of lab:
# Need a quiz
# 12 small tupperware containers during class
# TBS-T, ~300 ml / group for rinsing/washing blots
# 6 x 15 ul anti-p3 antibody (trying 1:1000 due to lack of anti-p3 material)
# 30 ml / group (1:1000 Goat-Anti-Mouse-Alkaline Phosphatase) in TBS-T + secondary antibody
# 25 ml / group developing solution for AP, 1 ml 25X developing solution (in 4&deg;C) + 24 ml MilliQ water, students add 250 ul of each of two solutions from kit in -20&deg;C
[[Image:Macintosh HD-Users-nkuldell-Desktop-p3Abinfo.jpg|thumb|p3 ab info]]


===[[20.109(F07): Module 1 oral presentations| Notes:Day8]]===
===[[20.109(F07): Module 2 oral presentations | Notes:Day 8]]===


==Recipes/Reagents==
==Recipes/Reagents==

Revision as of 04:33, 5 August 2007

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.
  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).

Day after lab:

  1. Change media on student's tranfection plates to 2 ml J1 growth media.

Notes:Day 3

Before lab:

Day of lab:

  1. Need quiz

Notes:Day 4

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

Notes: Day5

Notes:Day 6

Notes:Day 7

No prep. No quiz.

Notes:Day 8

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
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