20.109(F11): TA notes for module 2

Before module begins
Archive of TA notes Fall 2009 [[Media:TA NotebookGrading.jpg| 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 (can also spread plate with 25 ul Kan10 to check strain)
 * 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.

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 NB466 (= bacterial photography strain) on LB+Cam34+Amp25 (can spread with 25 ul Kan10 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 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 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

 * 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.
 * 1) 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.
 * 2) Amp: 25 mg/ml in H20. Filter and store at 4°. Use at 1:1000
 * 3) Cam: 34 mg/ml in EtOH. No need to filter sterilize. Use at 1:1000
 * 4) 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