20.109(S07): TA's notes for module 1

M13 Redesign Module

General notes

Before term begins:

1. Check that insert tag sequences make sense
2. Streak out NB251 (M13K07) and NB257 (M13K07 with PstI site) on LB+Kan. Make 10 overnight cultures (2-3 ml LB+Kan 1000:1) of each. Miniprep next day. Resuspend each pellet in 50 ul H20 and pool. Store at -20°. These will be used as cloning backbone starting on Day 1 of module.
3. Double-check volume of NO150/151 and NO154/155 for c-myc cloning.
4. NEB titers M13K07 on their strain ER2267 cat#E4103S. This strain is in the lab collection as NB271. Streak out on LB+Kan. The Kan is important to select for the F'.
5. Autoclave several racks of small and large test tubes.
6. Make 1L top agar, divide between autoclaved 250 ml bottles, 100 ml per bottle for reheating
7. Check volume/availability of needed kits, reagents:
   • M13K07 from NEB
   • restriction enzymes (PstI, ??)
   • Qiagen kit for agarose clean-up
   • T4 DNA ligase and buffer for ligation mix (NK will buy)
   • super competent XL1-blue cells
   • Miniprep solutions
   • Protein gels
   • Anti-myc antibody

Done: 2/1

• made 2L LB agar, poured plates
• 10 tubes each overnight of NB251 and NB257 in 2.5 ml LB+Kan (1:1000, so X ml LB + X ul Kan), inoculated with agarose plugs blown from capillary tubes

2/2

• miniprep plasmid DNA from overnights, 400ul of NB251, 550ul NB257 in water in -20 109 Plasmids box, also older tubes in tiny green box

Daily Notes

Notes:Day 1

Before lab:

1. Need to have backbone for restriction digest, made from miniprep of NB251 and NB257

Day of lab:

1. No quiz on day 1
2. Remember to freeze away digests (-20C) before leaving lab for the night.

Notes:Day 2

Before lab:

1. Need to set up 7x ER2267 (NB271) so there will be cells 1.2ml/group .
2. Need to pour 3 x 100 ml agarose gels for each day of lab, using one 10 well comb/gel.
3. Pour LB plates (2L), 6 plates per group, x 2 days
4. Double-check volume of NO150/151 and NO154/155 for c-myc cloning.
5. Check for phage stocks: M13K07 and another M13 phage called E4

Day of lab:

1. Need quiz for the students
2. Need Qiagen kit for gel purification. Do not put out any reagents that are incomplete (e.g., do not put out PE that does not have ethanol added to it).
3. Need 6 LB plates/group as well as 6 small sterile test tubes.
4. Put gels in boxes for running, remove combs
5. Need to melt 30 ml of top agar/group. Microwave in water bath for 2', invert to distribute any unmelted parts, heat another minute. It's very important that the top agar be fully melted for the students. Store molten in 55-60° water bath.

Notes:Day 3

Before lab:

1. Have LB-Kan plates poured. Need 5/group, 1L makes 30-40 plates, so need to make a couple liters.

Day of lab:

1. Need quiz
2. After lab, blow colony plugs into overnights for use on Day 4. Students will have labelled tubes.

Notes:Day 4

Before lab:

1. Aliquot solutions for miniprep (Solutions 1,2,3??) for each group so stocks don't get contaminated.

Day of lab:

1. Need quiz

Notes:Day 5

Day before lab:

1. Set up 7x NB271 for plaque assay
2. Pour LB plates
3. Protein gels

Day of lab:

1. Need quiz
2. 4 LB plates/group for plaque assay (no phage, + phage control, 2 different concentrations of precipitated supernatant)
3. Protein gels will have duplicate lanes so that blot can be cut in half. One half probed with anti-myc, the other anti-p3 or -p8 (new anti-p8, not tried yet). Figure out dilution of anti-p8??. Myc + control works.

Notes:Day 6

Day of lab:

1. Need quiz.
2. First drafts of papers due at beginning of class.
3. EHS rep will come in and talk about cell culture work during longer Western incubation period.
4. Talk about M13 refactoring during other incubation
5. Don't know if myc added is detectable??

p8 ab info

p3 ab info

Recipes/Reagents

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°.
2. Top Agar: 10 g Tryptone, 5 g Yeast Extract, 10 g NaCl, 1 g MgCl2*6H20 7 g Agar per liter. Autoclave then aliquot to 50 ml conical tubes or bottles. Store at RT. Melt in microwave in beaker of water, 2’ then keep molten in 55° water bath.
3. Amp: 100 mg/ml in H20. Filter and store at 4°. Use at 1:1000 in liq media. 2ml/L in plates
4. Tet: 20 mg/ml MeOH. Keep in dark at 4°. Use at 1:1000 in liq media. 1ml/L in plates
5. Kan: 25 mg/ml in H20. Filter and store at 4°. Use at 1:1000 in liq media. 2ml/L in plates.
6. 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
7. 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.
8. 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.
9. DNA gel: 1% agarose gel in 0.5X TBE, 1 g agarose in 100 ml, HOW MUCH EtBr?
10. Loading dye for agarose gel: 250 ul 1%XC (xylene cyanol), 750 ul 40% glycerol, 10 ul RNase. Store at RT.
11. 5X TBE 5.4% Tris base, 2.75% Boric Acid, 10mM EDTA, pH 8.0
12. 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% BPB
13. 1X sample dye for protein gel: 500 ul 2X sample dye, 400 ul H2O, 100 ul BME
14. TBS-T: Dilute 100 ml 10X TBS with 900 ml H20 then add 10 ml 10% Tween20
15. TBS-T +5% milk: add 2.5g milk powder to 50 ml TBS-T per blot.
16. Running Buffer: 1X TGS (10X from BioRad: 161-0772)
17. Transfer Buffer: 3.03 g Trizma base, 14.4g glycine, 200 ml MeOH to 1L with good H20. Store 4°.