Laboratory Fundamentals in Biological Engineering

TA's Notes for Module 1

General Notes

Notes: NB271 is a strain that can be selected for on LB-Kan media. It does not contain the M13KO7 plasmid, but it has the F pilus that is necessary for M13KO7 to infect it. NB251 is also a strain that can be selected for on LB-Kan- it contains the M13KO7 plasmid.

Before term begins:

1. Pour several liters of LB+Kan (~2L) and LB (~4L) plates.
2. Streak out NB251 (M13KO7) on LB+Kan. Make 16 overnight cultures (each 2.5 ml LB+Kan). Miniprep these for the vector DNA the next day. Resuspend each pellet in 50 µl H₂O. You do not have to pool since each group in the class will be given one tube for their cloning experiment that starts on Day1 of the module. Store at -20°.
3. Double-check volume of needed oligos: for cloning control, for sequencing.
4. NEB titers M13KO7 on their strain ER2267 cat#E4103S. This strain is in the lab collection as NB271. Streak out a plate on LB+Kan so you will have colonies to pick for liquid cultures before phage titration. The Kan is important to select for the F’. This is just to check that the phage stock is still active.
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:
   • M13KO7 from NEB
   • restriction enzymes (BamHI, others on list of no cutters (TXT))
   • Qiagen kit for agarose clean-up (need 1/pair of students)
   • T4 DNA ligase and buffer for ligation mix
   • super competent XL1-blue cells (need one tube of 200 µl/pair of students)
   • miniprep solutions
   • protein gels (need 3/lab section)
   • Anti-p3 antibody

Daily Notes

Day 1

Before lab:

1. Need to have plasmid for restriction digest, each group gest one miniprep of NB251

Day of lab:

1. No quiz on day 1
2. Keep cold:
   • PCR tubes with 10 µl BamH1
   • NEB buffer 2 on ice
3. Remember to freeze away digests (-20C) before leaving lab for the night

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 set up one overnight of 3 ml LB + Kan/group.
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 = 72 plates
4. Need top agar prepared
5. Check for phage stocks: M13KO7 and another M13 phage called E4
6. Check reagents and tubes in Qiagen Agarose Cleanup Kit
7. Need 1kb ladder and loading dye

Day of lab:

1. Need quiz (all quizzes 2 or 3 questions, 5 points)
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. 1 PCR tube/group
7. Need 6 LB plates/group taken out of -20C and 6 small sterile test tubes/group
8. 20 ml sterile water in a few 50ml conical tubes
9. Aliquot E4 and M13 phage stocks for dilutions, ~200 µl
10. 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).
11. Freeze purified backbone and annealed inserts, make sure all phage plates are in incubator.

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 15 ml falcon tubes

   • 100% ethanol, 6 x 15 ml falcon tubes

   • From -20°C freezer: 

     • students’ inserts and backbone
     • T4 DNA ligase, 3 aliquots in PCR tubes in cold-boxes (~10 µl each)
     • T4 ligase buffer, has ATP so must be kept cold (~50 µl in 3 eppendorfs)
     • BamHI, 2 aliquots in PCR tubes in cold-boxes (~10 µl each)
     • NEB Buffer 3 (~50 µl in 3 eppendorfs)
     • aliquots of yeast tRNA from 109 Antibodies box

   • only take out of -80°C freezer when first group is ready, competency decreases with time on ice 

     • super competent XL1-blue cells, one 200 µl aliquot per group

3. Sodium acetate 3M, 6 x 100 µl in microtubes

4. LB media, ~13 ml per group, put in 6 x 15 ml conical tubes

5. Sterile water, 6 x 1 ml in microtubes for ligation and resuspending DNA

6. LB-Kan plates out of 4C

7. M13KO7 plasmid DNA (mini-prepped before the beginning of term), 1:20 dilution for the positive control (~5ng)

8. Kanamycin, 10 µl per group (2 tubes of 50 µl), keep cold

9. large test tubes (4/group), 5 and 10 ml pipettes

10. ice buckets for competent cells

11. Streak NB276 (with teacher’s oligo) on LB-Kan plate to use in case students’ insert didn’t work

12. After incubating xformation plates for one day, blow colony plugs into overnights for use on Day 4. Students will have labelled tubes.

Day 4

Before lab:

1. Make 2L 1X TAE for gels and running buffer
2. Pour 3 agarose gels, 1% in TAE (100ml) + 2 µl EtBr each. Use 2 10-tooth combs/gel
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 µl Solution 1 (6 microtubes of 1000 µl)
     • 500 µl SDS 2% (6 microtubes of 1000 µl)
     • 500 µl 0.4M NaOH (6 microtubes of 1000 µl)
     • 600 µl Sol 3 (6 microtubes of 1000 µl)
     • 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

Day 5

Before lab:

1. If the volume of liquid culture of M13 infected E.coli is too small, add 1.5mL of LB-Kan to each tube and let grow at 37degreesC for 4-5hours max.

Day of lab:

1. Prepare reagents for sending DNA to sequencing facility
2. Oral presentation instructor will give talk during lab

Day 6

Day before lab:

1. Start overnights of 7x NB271 liquid cultures for plaque assay (3 ml of LB+Kan in large tubes), each group needs ~2 ml cells.
2. Start overnights of 7x NB251 liquid cultures for + control on Western (3 ml of LB+Kan in large tubes), each group needs ~1 ml.
3. Pour 2L of LB plates
4. Have protein gels and chambers
5. For each day, make microtubes of 1X sample buffer without BME (500 µl 2X + 400 µl H₂O) add 100 µl of BME to both on day of lab (could parafilm after adding BME, save 2 days). Need <1 ml / group.
6. Make 3 L of transfer buffer for each day and refrigerate
7. Make 1L 1X TBS-T in graduated cylinder, refrigerate, mix in 5% powdered milk close to lab day

Note: 2°Ab is goat antimouse (GAM-AP) in ab freezer box

Day of lab:

1. Need quiz
2. Turn on water baths and melt top agar
3. Protein gels and blot
   • Students’ candidates in liquid culture tubes
   • Blank for spec (900 µl H₂
   • Sample buffer + 100 µl BME
   • Lid locks for microtubes in hood
   • Boiling tank on hot plate in hood with boiling chips, be sure it’s turned off once students are done.
   • 2 protein gel chambers with 2 gels in one and 1 plus spacer in other, set up
   • “Kaleidoscope” protein molecular weight standard, in -20C in enzyme rack, aliquot 2x50 µl in microtubes, denature by boiling when students boil their samples
   • Running buffer, 1X TGS made when needed, 1L/chamber, 10X above sink
   • Transfer cassettes, ScotchBrite pads, filter paper, nitrocellulose
   • Western transfer buffer, 1L/tank so 3L/day
   • ice packs for Western
   • Blocking buffer TBS-T + 5% milk, 50 ml/group
   • Protein gels will have two groups/gel so that blot can be cut in half, and each probed with anti-p3
4. Plaque assay
   • top agar, melted in microwave and kept in 55C water bath
   • 200 µl x 10 /group NB271(=ER2267) bacteria for plaque assay
   • 10⁶ dilution of positive phage control in microtube
   • small sterile test tubes, 10/group
   • 10 LB plates/group for plaque assay (no phage, + phage control, 4 dilutions supernatant: 10⁰, 10², 10⁴, 10⁶, for each of two candidates)
   • 5 ml pipets and Pipetmen

Day 7

1. EHS rep will come in and talk about cell culture work during first Western incubation period
2. Talk about M13 refactoring during other incubation

Day of lab:

1. Need a quiz
2. 12 small tupperware containers during class
3. TBS-T, ~300 ml / group for rinsing/washing blots
4. 6 x 15 µl anti-p3 antibody (trying 1:1000 due to lack of anti-p3 material)
5. 30 ml / group (1:1000 Goat-Anti-Mouse-Alkaline Phosphatase) in TBS-T + secondary antibody
6. 25 ml / group developing solution for AP, 1 ml 25X developing solution (in 4°C) + 24 ml MilliQ water, students add 250 µl of each of two solutions from kit in -20°C

 

Image removed due to copyright restrictions. Product label for New England Biolabs Anti-M13 pIII Monoclonal Antibody (E8033S).

Recipes/Reagents

Growth media

1. LB: 10g Tryptone, 5g Yeast Extract, 10g 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-Kan plates, add the Kan after autoclaving, once the mixture has cooled down.
2. Top Agar: 10g Tryptone, 5g Yeast Extract, 10g NaCl, 1g MgCl2*6H₂O 7g 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 H₂O. 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 H₂O. Filter and store at 4°. Use at 1:1000 in liq media. 2ml/L in plates.

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 H₂O. Store at RT
2. Soln II for miniprep: equal parts 2% SDS (2g/100 ml H₂ O): 0.4M NaOH (1.6g/100 ml H₂O). Store components at RT. Mix just enough just before using.
3. Soln III for miniprep: 29.4 g KAc dissolved in 60 ml H₂O. 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 µl EtBr (wear nitrile gloves when handling EtBr!)
2. Loading dye for agarose gel: 250 µl 1% XC (xylene cyanol), 750 µl 40% glycerol, 10 µl RNase. Store at RT
3. 1kb marker: 10 µl 1kb marker stock (in -20 freezer), 10 µl loading dye, 90 µl H₂O
4. Western Blot
5. 5X TBE 5.4% Tris base, 2.75% Boric Acid, 10mM EDTA, pH 8.0
6. 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
7. 1X sample dye for protein gel: 500 µl 2X sample dye, 400 µl H₂
8. Running Buffer: 1X TGS (10X from BioRad: 161-0772)
9. Western Transfer Buffer: 3.03 g Trizma base, 14.4g glycine, 200 ml methanol to 1L with good H₂O. Store at 4°C. (25 mM Tris, 192 mM glycine, 20% v/v methanol)
10. TBS-T: Dilute 100 ml 10X TBS with 900 ml H₂O then add 10 ml 10% Tween20
11. TBS-T + 5% milk: add 2.5g milk powder to 50 ml TBS-T per blot