20.109(F11): TA notes for module 2
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Before module begins
Current module
Archive of TA notes
Fall 2009
Fall 2010,
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. If there is some problem with this "kinase dead" pair of strains, then it's also possible to use I566V mutant, NB469 and NB470.
- 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
- for 500 ml
- 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 30 ml of photography media/group. Best to make media in batches of 100 ml using 250 ml bottles with stir bars. Each bottle will provide enough for three groups. Pre-heat 42° waterbath!
- per 100 ml need:
- 0.5g Tryptone
- 0.25g Yeast Extract
- 0.5g NaCl (can use 100 ml of pre-made LB instead of these first 3 ingredients if there is lots of LB in the lab)
- 30mg Sgal
- 50mg ferric ammonium citrate
- 1 g Low Melting Point Agarose (be sure to use low melt point agar!)
- autoclave 30', stir at RT 3-5', cool to 42° in waterbath on instructor's bench at front of lab
- per 100 ml need:
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
- 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.
- 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.
- Amp: 25 mg/ml in H20. Filter and store at 4°. Use at 1:1000
- Cam: 34 mg/ml in EtOH. No need to filter sterilize. Use at 1:1000
- Kan: 10 mg/ml in H20. Filter and store at 4°. Use at 1:1000
DNA Miniprep
- 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
- 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.
- 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
- DNA gel: 1% agarose gel in 1X TAE, 1 g agarose, 100mL 1X TAE, 2 ul EtBr (wear nitrile gloves when handling EtBr!)
- Loading dye for agarose gel: 250 ul 1% XC (xylene cyanol), 750 ul 40% glycerol, 10 ul RNase. Store at RT.
- 1kb marker: 10uL 1kb marker stock (in -20 freezer), 10uL loading dye, 90uL H20
Western Blot
- 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
- 1X sample dye for protein gel using Sigma mix: 500 ul 2X sample dye, 200 ul H2O, 200 ul 10% SDS, 100 ul BME
- Transfer buffer: 3.03 g Trizma base, 14.4g glycine, 200 ml methanol, to 1L with good H2O. Store at 4°C
- 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