20.109(F10): TA notes for module 2

Before module begins

Archive of TA notes Fall 2009
Notebook grading checklist

Strains

• Streak out NB5 (= b-gal overproducing strain) on LB+Amp100
• Streak out NB188 (= strain for transformation/library screen) on LB+Amp200 (can also spread plate with 25 ul Kan25 to check strain)
• Streak out NB334 (= bacterial photography strain) on LB+Cam34+Amp200 (can also spread plate with 25 ul Kan25 to check strain)
• prepare electroporation competent cells of NB188. Innoculate 3x 50 ml LB+Amp100 with 250 ul of an overnight culture of NB188 that was grown in LB+Amp100. For F10: grown in 125 ml flask in room temp shaker starting at 5PM and harvested at 10AM next day. Spin in clinical centrifuge 3.2K 5' and then spin sup down again. Resuspend cells from each 50 ml culture in cold 10% glycerol and move to 10 epps (1 ml each). Pellet. 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 and freeze -80° <6 months. Test electroporation before module starts.

Materials

• check that K+ 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 NB334 (= bacterial photography strain) on LB+Cam34+Amp100 (can spread with 25 ul Kan25 to further check strain)
• 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 NB334 (one culture/group)--> 37° in LB+Cam34+Amp50+Kan25
• 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 building
• 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:

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

In advance

• Pour Tetrazolium+Cam34+Amp200 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

Day of lab

• Each pair needs ice bucket, electroporation cuvette, 2 Tetrazolium+Cam34+Amp200
• 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+ 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+ library
  • Before Day 5 of lab you will need to restreak and set up ONs

Day of lab

• NO quiz
• When students identify two candidate, you can restreak them onto LB+Cam34+Amp200 to grow 37° overnight.
• One day before the next lab you can set up overnight cultures in LB+Cam34+Amp100 in the dark.

Day 5

Lab has 2 parts:

• DNA: miniprep/digest/agarose gel/send to sequencing facility
• Protein Activity: b-gal

In advance

• ONE DAY BEFORE LAB: will need to set up overnight cultures of NB334 (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
• Pour 1 agarose gel, 1% in TAE (100ml) + 2 ul EtBr each. Use 2 10-tooth combs/gel.
• 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
• Midway through lab, assemble cocktail of NdeI and MluI in NEB3. For 20X cocktail, mix 250 ul H₂O, 50 ul 10X NEB3, 5 ul each enzyme. Add 15 ul of cocktail to 10 ul of student's samples, incubate 30 minutes at 37° and run on agarose gel with 1KB marker lane. Photograph and post.
• Near end of lab, thaw sequencing primer NO289 and dilute 1:100 in water. Each group needs about 13 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 (need to heat this to dissolve then filter sterilize) and 800 ul Amp100 + 400 ul Cam34. Let plates dry ON on bench and store in sleeves in 4°. Plates may be used for ~1 month.
3. Amp: 100 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

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