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

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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. 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
  5. MES cells and media
  6. Prep TC room
  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). Place each ml in cryotubes and wrap in paper towels, pack into styrofoam case and freeze -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.

Daily Notes

Notes:Day 1

Before lab:

  1. Need to have backbone for restriction digest, each group gest one miniprep of NB251

Day of lab:

  1. No quiz on day 1
  2. Keep cold:
    • PCR tubes with 10 ul BamH1
    • NEB buffer 2 on ice
  3. Remember to freeze away digests (-20C) before leaving lab for the night.

Notes:Day 2

Before lab:

  1. Need to set up ER2267 (NB271) so there will be cells for 1.2 ml/group. Plate on LB+Kan, then set up one overnight of 3 ml LB + Kan/group.
  2. Need to pour 3 x 100 ml agarose gels for each day of lab, using one 10-well comb (thicker teeth) in each gel.
  3. Pour LB plates (2L), 6 plates per group, x 2 days
  4. Need top agar prepared
  5. Check for phage stocks: M13K07 and another M13 phage called E4
  6. Check reagents and tubes in PCR Cleanup Kit
  7. Need 1kb ladder and loading dye

Day of lab:

  1. Need quiz (all quizzes 2 or 3 questions, 5 points).
  2. Put gels in boxes for running, remove combs, add ~500 ml 1X TAE
  3. Need to melt 30 ml of top agar/group. Microwave in water bath for 2', invert to distribute any unmelted parts, heat another minute. (SC: I microwaved 1', swirled, microwaved 30", and it was melted, then into 55C water bath) It's very important that the top agar be fully melted for the students. Store molten in 55-60°C water bath.
  4. Bacterial overnights out
  5. Pipetmen, 5 ml pipettes and 50C water baths for phage plating
  6. 1 PCR tube/group
  7. Need 6 LB plates/group taken out of -20C and 6 small sterile test tubes/group.
  8. 20 ml sterile water in a few 50ml conical tubes
  9. Aliquot E4 and M13 phage stocks for dilutions, ~200 ul
  10. Need Qiagen kit for gel cleanup. Do not put out any reagents that are incomplete (e.g., do not put out PE that does not have ethanol added to it).
  11. Freeze purified backbone and annealed inserts, make sure all phage plates are in incubator.

Notes:Day 3

Before lab:

  1. Have LB-Kan plates poured. Need 5/group = 60, 1L makes 30-40 plates, so need to make a couple liters.
  2. Refill burners and jars with 100% ethanol

Day of lab:

  1. Need quiz
  2. 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
  3. Sodium acetate 3M, 6 x 100 ul in microtubes
  4. LB media, 10 ml per group, put in 50 ml conical tubes (3 x 30)
  5. Sterile water, 6 x 1 ml in microtubes for ligation and resuspending DNA
  6. LB-Kan plates out of 4C
  7. Kanamycin, 10 ul per group (2 tubes of 50 ul), keep cold
  8. large test tubes (4/group), 5 and 10 ml pipettes
  9. ice buckets for competent cells
  10. After incubating xformation plates for one day, blow colony plugs into overnights for use on Day 4. Students will have labelled tubes.

Notes:Day 4

Before lab:

  1. Make 2L 1X TAE for gels and running buffer
  2. Pour 3 agarose gels, 1% in TAE (100ml) + 2 ul EtBr each. Use 2 10-tooth combs/gel.
  3. Aliquot solutions (see below)

Day of lab:

  1. Need quiz
  2. Aliquot solutions for miniprep (Solutions 1, 3, NaOH and SDS) for each group so stocks don't get contaminated.
    • Each group needs:
      • 400 ul Solution 1 (6 microtubes of 1000 ul)
      • 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
  3. Keep cold:
    • buffers
    • restriction enzymes
  4. ice buckets (one per bench should be fine, just for miniprep)
  5. 1 ml serological pipets for alcohols (optional)
  6. loading dye for gels

Notes:Day 5

Notes:Day 6

Day before lab:

  1. Start overnights of 7x NB271 liquid cultures for plaque assay (3 ml of LB+Kan in large tubes), each group needs ~2 ml cells.
  2. Start overnights of 7x NB251 liquid cultures for + control on Western (3 ml of LB+Kan in large tubes), each group needs ~1 ml.
  3. Pour 2L of LB plates
  4. Have protein gels and chambers
  5. 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.
  6. Make 3 L of transfer buffer for each day and refrigerate
  7. 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

Day of lab:

  • Need quiz
  • Turn on water baths and melt top agar
  • Protein gels and blot
  1. Students' candidates in liquid culture tubes
  2. Blank for spec (900 ul H2O and 100 ul LB)
  3. Sample buffer + 100 ul BME
  4. Lid locks for microtubes in hood
  5. Boiling tank on hot plate in hood with boiling chips, be sure it's turned off once students are done.
  6. 2 protein gel chambers with 2 gels in one and 1 plus spacer in other, set up
  7. "Kaleidoscope" protein molecular weight standard, in -20C in enzyme rack, aliquot 2x50 ul in microtubes, denature by boiling when students boil their samples
  8. Running buffer, 1X TGS made when needed, 1L/chamber, 10X above sink
  9. Transfer cassettes, ScotchBrite pads, filter paper, nitrocellulose
  10. Western transfer buffer, 1L/tank so 3L/day
  11. ice packs for Western
  12. Blocking buffer TBS-T + 5% milk, 50 ml/group
  13. Protein gels will have two groups/gel so that blot can be cut in half, and each probed with anti-p3.
  • Plaque assay
  1. top agar, melted in microwave and kept in 55C water bath
  2. 200 ul x 10 /group NB271(=ER2267) bacteria for plaque assay
  3. 10^6 dilution of positive phage control in microtube
  4. small sterile test tubes, 10/group
  5. 10 LB plates/group for plaque assay (no phage, + phage control, 4 dilutions supernatant: 10^0,2,4,6, for each of two candidates)
  6. 5 ml pipets and Pipetmen

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:

  1. Need a quiz
  2. 12 small tupperware containers during class
  3. TBS-T, ~300 ml / group for rinsing/washing blots
  4. 6 x 15 ul anti-p3 antibody (trying 1:1000 due to lack of anti-p3 material)
  5. 30 ml / group (1:1000 Goat-Anti-Mouse-Alkaline Phosphatase) in TBS-T + secondary antibody
  6. 25 ml / group developing solution for AP, 1 ml 25X developing solution (in 4°C) + 24 ml MilliQ water, students add 250 ul of each of two solutions from kit in -20°C
p3 ab info

Notes:Day8

Recipes/Reagents

  1. 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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