20.109(F11): TA notes for module 2

Before module begins

Current module
Archive of TA notes
Fall 2009
Fall 2010,

Notebook grading checklist

Strains

• Streak out NB5 (= b-gal overproducing strain) on LB+Amp100
• Streak out NB462 (= strain for transformation/library screen) on LB+Amp25 (can also spread plate with 25 ul Kan10 to check strain)
• Streak out NB466 (= bacterial photography strain, alternative = NB334) on LB+Cam34+Amp25+Kan10
• Streak out NB467 and NB468 (= kinase dead strain H557A, in XL1-blue and photography strain respectively). NB467 should be streaked out on LB+Cam34 and NB468 on LB+Amp25+Cam34+Kan10.
• prepare electroporation competent cells of NB462. Innoculate 3x 50 ml LB+Amp25+Kan10 with 50 ul of an overnight culture of NB462 that was grown in LB+Amp25+Kan10. For F11: grown in 125 ml flask in room temp shaker starting at 3PM and harvested at 9AM next day. Pool cultures in one flask to make uniform (measured OD600 = 0.515). Divide between 4 50 ml conical tubes of ~35 ml each to spin in clinical centrifuge 3.2K 5' and then spin sup down again. Resuspend cells from each 50 ml conical in 10 ml cold 10% glycerol (40 ml total) and move 20 ml to 20 epps (1 ml each). Pellet. Add other 20 ml to those epps. Pellet again. Wash cells with 1 ml cold 10% glycerol. Pellet. Wash cells with 250 ul cold 10% glycerol. Resuspend cells in 50 ul cold 10% glycerol for a total of 20 epps of EP comp tubes and freeze -80° <6 months. Test electroporation before module starts.
• miniprep H557A kinase dead control plasmid from NB467 and/or I566V mutant from NB469. These will be used for electroporation/screening controls on Day 3.

Materials

• check that K-P+ library is available
• check stocks for protein gels/western including anti-H6EnvZ antibody, Epicentre prot ext'n soln, detection kit, kaleidoscope markers, gels, buffers

Day 1

Lab has 3 parts:

• Test in Solid Media
• Test in Liquid Media
• Practice b-gal

In advance of lab

• Streak out strains NB5 (= b-gal overproducing strain) on LB+Amp100
• Streak out NB466 (= bacterial photography strain) on LB+Cam34+Amp25+Kan10
• Autoclave large and small tubes if needed

Day before lab

• Set up 2.5 ml overnights of NB5 (one culture/group)-->37° in LB+Amp100
• Set up 2.5 ml overnights of NB466 (one culture/group)--> 37° in LB+Cam34+Amp25+Kan10
• Make at least 500 ml of Z-buffer
  • for 500 ml
    • 8.05 g Na₂HPO₄*7H₂0
    • 2.75 g NaH₂PO₄*H₂O
    • 0.375 g KCl
    • 0.123 g MgSO₄*7H₂0
  • Dissolved in 500 ml H₂0 final volume
• Make 100 ml 4 mg/ml ONPG in Z-buffer, aliquot 1 ml/group and freeze rest
• Make 250 ml 1 M Na2CO3 in water, aliquot 5 ml/group and leave rest at RT
• Make 0.1% SDS (can dilute 10% solution), aliquot 0.5 ml/group and leave rest at RT

Day of lab

• One box of cuvettes/team
• Sleeve of empty petri dishes (at least 2 needed per team)
• Autoclave 50 ml of photography media/group. Pre-weigh into a 1 liter bottle, autoclave 30 min over lunch (add stirbar before autoclaving)! Stir, then aliquot into 15 ml falcon tubes, keeping falcon tubes in a 42 degree waterbath on instructors bench. Pre-heat waterbath! Students can then take the 15 ml conical tubes when they need them.
  • per 50 ml need:
    • 0.5g Tryptone
    • 0.25g Yeast Extract
    • 0.5g NaCl
    • 15mg Sgal
    • 25mg ferric ammonium citrate
    • 0.5g Low Melting Point Agarose (be sure to use low melt point agar!)
  • autoclave 30', cool to 42° in waterbath on instructor's bench at front of lab

Day 2

Lab has 4 parts:

• Light/Dark b-gal
• Bacterial Photograph
• TinkerCell Model and Simulation
• Oral Presentation Instruction in lab

Day of lab

• see day 1 info except don't need NB5 overnight cultures
• also need quiz

Day 3

Lab had 4 parts:

• Electroporate Library
• Registry of Std Biological Parts
• TinkerCell Simulations
• Electronics

In advance

• Pour Tetrazolium+Cam34+Amp25+Kan10 petri dishes, need 2/group.
• Check stock of cuvettes, SOC media
• pre-run electroporation to check on best volume of cells for students to plate
• Miniprep kinase dead mutants to electroporate as controls

Day of lab

• Each pair needs ice bucket, electroporation cuvette, 2 Tetrazolium+Cam34+Amp25+Kan10
• Front bench needs at least 2x electronics lab set up (see protocols)
• Gel running bench can have 2 electroporation machines set up
• In ice bucket on front bench: thaw K-P+ library, EP cells just before use
• Front bench also needs alcohol burners, EtOH beakers, spreaders, strikers

Day 4

• Students will identify 2 candidates from K-P+ library.
  • TA needs to restreak for single colonies on the first day between labs, then
  • set up 2.5 ml overnights in light and dark from single colony on the second night between labs.
    

This is a lot of work!

Day of lab

• NO quiz
• When students identify two candidate, you can restreak them onto LB+Cam34+Amp25+Kan10 to grow 37° overnight.
• One day before the next lab you can set up overnight cultures in LB+Cam34+Amp25+Kan10 in the light and dark. Will also want to set up unmutated and kinase dead controls (NB466 and NB468 and/or NB470)

Day 5

Lab has 3 parts:

• DNA: miniprep/send to sequencing facility
• Protein Activity: b-gal from light and dark
• Writing instruction

In advance

• ONE DAY BEFORE LAB: will need to set up overnight cultures of NB466 (bacterial photography strain) and the 2 mutants that the students have selected. A 5 ml overnight in LB+Cam34+Amp100 for each should be enough.
• aliquot miniprep solutions (see below)

Day of lab

• Need quiz
• Each group needs miniprep solutions (Solutions 1, 3, NaOH and SDS) for each group so stocks don't get contaminated.
  • 400 ul Solution I
  • 500 ul SDS 2%
  • 500 ul 0.4M NaOH
  • 600 ul Solution III
  • 4 ml 100% Ethanol
  • 2 ml 70% Ethanol
  • sterile water bottle for each group
• Near end of lab, thaw sequencing primer NO296 and dilute 1:20 in water. Each group needs about 10 ul of dilute oligo. Also need a few 8 strip PCR tubes to send to seq.
• Each group also needs b-gal assay solutions, like Day 1 of the module.
  • Z-buffer, 5 ml/ group
  • 4 mg/ml ONPG in Z-buffer, aliquot of 1 ml/group
  • 1 M Na2CO3 in water, aliquot of 5 ml/group
  • 0.1% SDS in water, aliquot of 0.5 ml/group
  • Box of cuvettes

End of lab

• Store remainder of overnight cultures for Day 6 protein gel
• Some students may want to set up dark/light overnight liquid cultures from their samples
• Post image of agarose gel
• Run samples to sequencing facility

Day 6

• Photograph
• Seq analysis
• Protein Gel/blot

In advance of lab

• check levels on solutions for protein gel (TGS, TBS, Tween, milk powder)
• make Transfer Buffer and store in delicase (4°)
  • 3.03 g Trizma base
  • 14.4g glycine
  • 200 ml methanol
  • to 1L with good H₂O
  • Store at 4°C

Day of lab

• Need quiz
• Prepare 2X SB
  • 500 ul Sigma dye G2526
  • 200 ul H2O
  • 200 ul 10% SDS
  • 100 ul BME
• Dilute purified H6-EnvZ protein and mix with 2xSB (2.5 ul into 100 ul 2xSB). Students will need 40 ul/team so aliquot 50ul for each.
• Make 800 ul of Epicentre "EasyLyse" solution for each group. This is done by mixing (in the following order):
  • 0.4 ml sterile H₂O
  • 1.6 ul 1M MgCls (kept in Epicentre kit at RT)
  • 0.4 ml Lysis Buffer (kept in Epicentre kit at RT)
  • 0.8 ul "enzyme mix" (kept in tote in -20°)
  • mix just before lab and leave on ice until students request it
• Make TBS+T+milk (25 ml/group)
  • TBS-T: Dilute 100 ml 10X TBS with 900 ml H20 then add 10 ml 10% Tween20
  • TBS-T + 5% milk: add 2.5g milk powder to 50 ml TBS-T. Mix on stir plate or in conical at 37° on nutator until milk dissolved
  • Transfer buffer (1 liter /tank). One tank holds two gels, i.e. 4 groups.
• Set up acyrlamide gel in 1X TGS just before lab

Day 7

• Probe Western
• Need quiz

In lab

• Bring blots from fridge to RT just before lab starts
• Just before lab thaw primary antibody to His6-EnvZ. You will need 10 ul per group.
• Make sure shaker platform in chemical hood is set to rotate at ~60 rpm.
• Confirm that there is sufficient GAR-AP antibody (4° deli case, from Sigma)
• Student groups will need ~250 ml TBS-T each.
• Aliquot 1X development solution so 25 ml conical available for each group.
• Bring out development kit just as needed.
• Once gels developed, scan or photograph and post images to talk page of today's lab.

Day 8

• No quiz
• "only" Journal Club

Recipes/Reagents

Growth media

1. LB: 10 g Tryptone, 5 g Yeast Extract, 10 g NaCl per liter. 20g of Agar for plates. Autoclave 30 minutes with stirbar. Pour when ~55°. Let plates dry ON on bench and store in sleeves in 4°. For LB+ antibiotics plates, add the antibiotics after autoclaving, once the mixture has cooled down.
2. Tetrazolium indicator media: for 400 ml: 10.2 grams Antibiotic Medium #2, 20 mg Tetrazolium (kept in 4° delicase with chemicals). Add 380 ml H₂O then heat at setting "5" in hood on stirplate to help dissolve agar. Autoclave 30 minutes and cool to ~55° then add 20 ml of 20% lactose (4g/20 ml H20, need to heat this to dissolve then filter sterilize) and 400 ul Amp25 + 400 ul Cam34 + 400 ul Kan10. Let plates dry ON on bench and store in sleeves in 4°. Plates may be used for ~1 month.
3. Amp: 25 mg/ml in H20. Filter and store at 4°. Use at 1:1000
4. Cam: 34 mg/ml in EtOH. No need to filter sterilize. Use at 1:1000
5. Kan: 10 mg/ml in H20. Filter and store at 4°. Use at 1:1000

DNA Miniprep

1. Soln I for miniprep: 2.3 ml 40% glucose, 2.5 ml 1M Tris 8, 2 ml 0.5M EDTA. To 100 ml with good H20. Store at RT
2. Soln II for miniprep: equal parts 2% SDS (2g/100 ml H20): 0.4M NaOH (1.6g/100 ml H20). Store components at RT. Mix just enough just before using.
3. Soln III for miniprep: 29.4 g KAc dissolved in 60 ml H20. Add 11.5 ml glacial acetic acid. Bring to 100 ml final volume. Store at RT.

Agarose Gel

1. DNA gel: 1% agarose gel in 1X TAE, 1 g agarose, 100mL 1X TAE, 2 ul EtBr (wear nitrile gloves when handling EtBr!)
2. Loading dye for agarose gel: 250 ul 1% XC (xylene cyanol), 750 ul 40% glycerol, 10 ul RNase. Store at RT.
3. 1kb marker: 10uL 1kb marker stock (in -20 freezer), 10uL loading dye, 90uL H20

Western Blot

1. 2X sample dye for protein gel (no BME): 4 ml 10% SDS, 5 ml 40% glycerol, 1 ml 1M Tris 6.8, 0.5 ml <1% bromophenol blue, stocks on NK's bench
2. 1X sample dye for protein gel using Sigma mix: 500 ul 2X sample dye, 200 ul H2O, 200 ul 10% SDS, 100 ul BME
3. Transfer buffer: 3.03 g Trizma base, 14.4g glycine, 200 ml methanol, to 1L with good H2O. Store at 4°C
4. TBS-T: Dilute 100 ml 10X TBS with 900 ml H20 then add 10 ml 10% Tween20
5. TBS-T + 5% milk: add 2.5g milk powder to 50 ml TBS-T. Mix on stir plate or in conical at 37° on nutator until milk dissolved