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

M13 Redesign Module

20.109

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 or 20 of NB257 since it works for either BamHI or PstI. Miniprep these for the vector DNA the 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
   • super competent XL1-blue cells
   • Miniprep solutions
   • Protein gels
   • Anti-myc antibody

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. Keep cold:
   • PCR tubes with 10 ul BamH1, PstI
   • NEB buffers 2,3 on ice
3. Remember to freeze away digests (-20C) before leaving lab for the night.

Notes:Day 2

Before lab:

1. Need to set up ER2267 (NB271) so there will be cells for 1.2 ml/group. Plate on LB+Kan, then overnight 3 ml LB + Kan in tubes.
2. Need to pour 3 x 100 ml agarose gels for each day of lab, using one 10-well comb (thicker teeth) in each gel.
3. Pour LB plates (2L), 6 plates per group, x 2 days
4. Need top agar prepared
5. Double-check volume of NO150/151 and NO154/155 for c-myc cloning.
6. Check for phage stocks: M13K07 and another M13 phage called E4
7. Check reagents and tubes in PCR Cleanup Kit
8. Need 1kb ladder and loading dye

Day of lab:

1. Need quiz
2. Put gels in boxes for running, remove combs, add ~500 ml 1X TAE
3. Need to melt 30 ml of top agar/group. Microwave in water bath for 2', invert to distribute any unmelted parts, heat another minute. (SC: I microwaved 1', swirled, microwaved 30", and it was melted, then into 55C water bath) It's very important that the top agar be fully melted for the students. Store molten in 55-60°C water bath.
4. Bacterial overnights out
5. Pipetmen, 5 ml pipettes and 50C water baths for phage plating
6. Mix up equal volumes of oligos for gel control, then load 5 ul + 0.5 ul loading buffer in one lane of each gel
7. 1 PCR tube/group
8. Need 6 LB plates/group taken out of -20C and 6 small sterile test tubes/group.
9. 20 ml sterile water in a few 50ml conical tubes
10. Aliquot E4 and M13 phage stocks for dilutions, ~200 ul
11. Need Qiagen kit for gel cleanup. Do not put out any reagents that are incomplete (e.g., do not put out PE that does not have ethanol added to it).
12. Freeze purified backbone and annealed inserts, make sure all phage plates are in incubator.

Notes:Day 3

Before lab:

1. Have LB-Kan plates poured. Need 5/group = 60, 1L makes 30-40 plates, so need to make a couple liters.
2. Refill burners and jars with 100% ethanol

Day of lab:

1. Need quiz
2. Keep cold
   • 70% ethanol, 6 x 1.5 ml (full microtubes)
   • 100% ethanol, 6 x 1 ml
   • From -20°C freezer:
     • students' inserts and backbone
     • T4 DNA ligase, 3 aliquots in PCR tubes in cold-boxes
     • T4 ligase buffer, has ATP so must be kept cold
     • BamHI, PstI, 2 aliquots in PCR tubes in cold-boxes
     • NEB Buffers 2,3
     • aliquots of yeast tRNA from 109 Antibodies box
   • only take out of -80 freezer when first group is ready, competency decreases with time on ice
     • competent cells, one 200 ul aliquot per group
3. Sodium acetate 3M, 6 x 100 ul in microtubes
4. LB media, 10 ml per group, put in 50 ml conical tubes (3 x 30)
5. Sterile water, 6 x 1 ml in microtubes for ligation and resuspending DNA
6. LB-Kan plates out of 4C
7. Kanamycin, 10 ul per group (2 tubes of 50 ul), keep cold
8. large test tubes (4/group), 5 and 10 ml pipettes
9. ice buckets for competent cells
10. After incubating xformation plates for one day, blow colony plugs into overnights for use on Day 4. Students will have labelled tubes.

Notes:Day 4

Before lab:

1. Make 2L 1X TAE for gels and running buffer
2. Pour 3 1% TAE gels + EtBr 100ml with 2 10-tooth combs
3. Aliquot solutions (see below)

Day of lab:

1. Need quiz
2. Aliquot solutions for miniprep (Solutions 1, 3, NaOH and SDS) for each group so stocks don't get contaminated.
   • Each group needs:
     • 400 ul Solution 1 (6 microtubes of 1000 ul)
     • 500 ul SDS 2% (6 microtubes of 1000 ul)
     • 500 ul 0.4M NaOH (6 microtubes of 1000 ul)
     • 600 ul Sol 3 (6 microtubes of 1000 ul)
     • 4 ml 100% Ethanol (6 x full 15 ml tubes)
     • 2 ml 70% Ethanol (6 x ~10 ml in 15 ml tubes)
     • sterile water bottle for each group
3. Keep cold:
   • buffers
   • restriction enzymes
4. ice buckets (one per bench should be fine, just for miniprep)
5. 1 ml serological pipets for alcohols (optional)
6. loading dye for gels

Notes:Day 5

Day before lab:

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

Day of lab:

1. Need quiz

• protein gels and blot

1. Sample buffer: glycerol, SDS, BME, bromophenol blue
2. Lid locks for microtubes
3. Boiling method
4. Protein gels set up
5. "Kaleidoscope" protein molecular weight standard
6. Transfer cassettes, ScotchBrite pads, filter paper, nitrocellulose
7. transfer buffer
8. Blocking buffer TBS-T + 5% milk
9. 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.

• plaque assay

1. 25% PEG 8000 in 2.5M NaCl, 166 ul/group
2. sterile water
3. ice bucket for each bench
4. top agar, melted in microwave and kept in 55C water bath
5. bacteria for plaque assay?
6. 4 LB plates/group for plaque assay (no phage, + phage control, 2 different concentrations of precipitated supernatant)

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 1X TAE, 1 g agarose, 10 ml 10X TAE, 90 ml water, 2 ul 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 methanol to 1L with good H20. Store at 4°. (25 mM Tris, 192 mM glycine, 20% v/v methanol)