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 and NB257 on LB+Kan. One is M13K07 with PstI site and other is standard M13?? Qiagen miniprep 10 overnights of each. 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. Streak out NB6 (XL1-blue) on LB spread with 25 ul Tet. The tet is important to select for the F'
5. NEB titers M13K07 on their strain ER2267 cat#E4103S. This strain is in the lab collection as NB271. Streak out on LB+Kan.
6. Autoclave several racks of small and large test tubes.
7. Check volume/availability of needed kits, reagents:
   • M13K07 from NEB
   • Qiagen kit for agarose clean-up
   • T4 DNA ligase and buffer for ligation mix
   • super competent XL1-blue cells
   • restriction enzymes (PstI, ??)

Daily Notes

Notes:M1D1

1. No quiz on day 1
2. Remember to freeze away digests before leaving lab for the night.

Notes:M1D2

Day before lab:

1. Need to set up 7x ER2267 (NB271).

Day of lab:

1. Need quiz for the students
2. Need to pour 3 x 100 ml gels for each day of lab, using one 10 well comb/gel.
3. Need Q-kit for gel purification. Do not put out any reagents that are incomplete (e.g. do not put out PE that has not ethanol added to it).
4. Need 6 LB plates/group as well as 6 small sterile tubes.
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:M1D5

Day before lab:

1. Set up 7XNB271 for plaque assay

Day of lab:

1. 4 LB plates/group for plaque assay (no phage, + phage control, 2 conc of ppt sup)

Notes:M1D6

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. Loading dye for agarose gel:250 ul 1%XC, 750 ul 40% glycerol, 10 ul RNase. Store at RT.
10. 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
11. 1X sample dye for protein gel: 500 ul 2X sample dye, 400 ul H2O, 100 ul BME
12. TBS-T: Dilute 100 ml 10X TBS with 900 ml H20 then add 10 ml 10% Tween20
13. TBS-T +5% milk: add 2.5g milk powder to 50 ml TBS-T per blot.
14. Running Buffer: 1X TGS (10X from BioRad: 161-0772)
15. Transfer Buffer: 3.03 g Trizma base, 14.4g glycine, 200 ml MeOH to 1L with good H20. Store 4°.