20.109(S08): TA notes for module 1

General notes

Key preparation:

• In case something goes wrong, spare PCR product should be prepared by TA prior to term.
• A plan for preparing the high volume of cell culture must be determined well in advance.

Plasmids used:

• pCX-EGFP:
  • contains full-length EGFP gene
  • Amp resistant
  • f1 ori
  • SV40 ori

• pCX-NNX:
  • a mock version of pCX-EGFP, contains no EGFP gene
  • Amp resistant
  • f1 ori
  • some altered restriction sites compared to pCX-EGFP, and SV40 ori deleted

• pCX-EGFP with 3' deletion - still have this in lab, how labeled?

Daily Notes

Day 1

Materials required:

1. PCR Master Mix (2.5X), ~ 50 μL per group
2. DI water, prep 100 μL aliquot per group
3. 5 μL aliquots each of pCX-EGFP, D32N-fwd, D32N-rev
4. 2 PCR tubes per group

Keep everything cold!

Day of Lab:

• Lab orientation quiz (not a TA quiz) will be taken today.
• Remember to freeze PCR products when they are ready.

Instructor's Bench

• Ice bucket
• PCR Master Mix (taken out of -20C midway through labtime)
• Sterile H20 in 15mL Falcon
• Primters for PCR (D32N-fwd: NO47, D32N-rev: NO48, 100 pmol/ul)
• Template for PCR (pCX-EGFP 100 ng/ul)
• Beaker with PCR tubes

Prep Work

pCX-EGFP has to be prepared from the glycerol stock. The bacterial stock is NBXXX and a few Qiagen minipreps should be enough for the module (some will also be needed later as a transfection control). You should determine the concentration of the stock. For PCR, dilute stock to about 100 ng/ul.

Other DNA samples that will be needed later in the module come from:

• NBXXXX pCX-NNX
• NB153 pCX-D32N (Xba/RI)
• NB168 pCX-D3G+Linker -- This plasmid needs to be cut in three ways and each bkb purified!

Check the PCR machine has proper protocol under NK1: 94C 4min

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94C 1min /n 55C 1min (Repeat 35 times) 72C 1min

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72C 10min 4C hold forever

Day 2

Materials required:

1. Qiagen QIAquick PCR purification kit
   • order # 28104, 50 rxns
   • 1 rxn per group
2. pCX-NNX, 10 μL per group
3. NEB2 Buffer (~5 μL used per group)
4. EcoRI and XbaI enzymes (~2 μL used per group)

Day of Lab:

• Quiz (prepared by TA).
• Thaw PCR products and template on ice.

Day 3

Materials required:

1. Qiagen QIAquick gel extraction kit
   • 2 extractions per group
   • order # 28704, 50 rxns
2. isopropanol (prep 500 μL aliquots)
3. sterile DI water (prep 500 μL aliquots)
4. loading dye for agarose gel electrophoresis (prep 35 μL aliquots)
5. 1% agarose gels prepared in 0.5X TBE buffer
   • each group has 10 samples, so requires 10 wells
   • prepare 1 gel per two groups, but with 2 combs
   • also prepare 1 more gel (2 combs!), for running purified products
6. Single-enzyme digests - may be prepared on Day 2.
   • pCX-NNX, XbaI cut only
   • pCX-NNX, EcoRI cut only
   • large (400 μL) and small (25 μL) volume reactions should be done for each of the above

Day of Lab:

• Quiz (prepared by TA).
• Run purified products (2 per group) on an agarose gel at the end of the day - post photograph.
• Freeze DNA at end of day.

Day 4

Materials required:

1. Retrieve at last minute and keep on cold rack: ligation buffer, T4 ligase
2. 3 M sodium acetate (prep 100 μL aliquots)
3. Yeast tRNA (prep 25 μL aliquots)
4. Cold 100% ethanol (prep 1 mL aliquots)
5. Cold 70% ethanol (prep 3 mL aliquots)
6. Sterile DI water (prep 100 μL aliquots)
7. pCX-EGFP (1 μL/50 ng per group)
8. competent cells
   • XL1-Blue from Stratagene, cat # ?200249?
   • 1 tube per group
9. LB+Amp plates, 5 per group + spares
   • VWR cat. #
   • may get ~ 40 plates/Liter LB
10. LB liquid medium

Day of Lab:

• Quiz (prepared by TA).
• Keep reagents for ligation reactions cold, make available to students as needed.
• Demonstrate and supervise bacterial transformation protocol.
• Transform cells with pCX-NNX to get fresh colonies.
  • May be done a week or so in advance, if desired.
  • Can also just try streaking frozen stock, if available.
• Tomorrow pick colonies for inoculations - 3 candidates per group, plus 1 pCX-NNX.

Day 5

Materials required:

• Liquid cultures (3 candidates, 1 pCX-NNX control per group).
• Miniprep solutions
  • Soln I (prep 400 μL aliquots)
  • Soln II components (prep 600 μL aliquots, each)
  • Soln III (prep 800 μL aliquots)
  • 100 % ethanol (prep 5 mL aliquots)
  • 70 % ethanol (prep 3 mL aliquots)
  • Sterile DI water (prep 250 μL aliquots)
• Digests: have all 4 NEB buffers and many restriction enzymes available

Day of Lab:

• Quiz (prepared by TA).
• Ensure DNA is frozen at end of day.

Day 6

Long-term preparation:

• Per student, one 60 mm dish of MES cells near confluence

Materials required:

1. Agarose gels
   • Half of class at a time will be in TC, so can't prepare just 1 gel per two groups
   • Probably want to prepare 4 gels per 6 groups, but can prepare 1 per group for simplicity
2. TC reagents
   • Aliquot each at 10-20% excess
   • PBS (12 mL per group)
   • autoclaved gelatin (6 mL per group)
   • Trypsin (1 mL per group)
   • J1 medium(three tubes per group with precisely 10 mL each)

Day of Lab:

• Quiz (prepared by TA).

Day 7

Long-term preparation:

• Per group, one 24-well plate with 16 seeded wells.
  • Seed with 10⁵ cells 24 hrs prior to use.
• Need plasmid with EGFP truncated at 3' end.

Materials required:

1. Lipofectamine (prep 50 μL aliquots)
2. OptiMEM (prep 2 mL aliquots)
3. TC reagents
   • PBS (10 mL per group)
   • T'xn Medium (10 mL per group)

Day of Lab:

• Quiz (prepared by TA).

Day 8

Materials required:

1. TC reagents
   • PBS (20 mL per group)
   • Trypsin (4 mL per group)
   • OptiMEM (2 mL per group) - 100 μL seems low for FACS
2. FACS tubes
   • 16 per group, plus extras
   • BD Falcon tubes only, VWR cat # 60819-310

Day of Lab:

• No quiz.
• TA will run flow cytometer, and faculty will guide student preparations in the lab, or vice-versa.

Recipes/Reagents

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!)
   • 0.5X TBE insteadof 1X TAE for this lab... ?
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 °C freezer), 10 μL loading dye, 90 μL H20

Bacterial growth media

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°. For LB-Amp plates, add the Amp after autoclaving, once the mixture has cooled down.
2. Amp: 100 mg/ml in H20. Filter and store at 4°. Use at 1:1000 in liq media. 2ml/L in plates
3. 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.

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 H20. Store at RT
2. 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.
3. 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.

Mammalian cell culture

• JI Growth Media: 500 ml DMEM (high glucose), 50 ml FBS (Atlanta Biologic, Inc.), 5 ml P/S/G, 1 ml BME, 5 ml NEAA. Filter then add 50 ul LIF. Store 4°C
• Pre-Transformation Media: 500 ml DMEM (high glucose), 50 ml FBS (Atlanta Biologic, Inc.), 5 ml 100XG, 1 ml BME, 5 ml NEAA. Filter then add 50 ul LIF.
• J1 culture medium
  • 100 U/ml Pen/Strep
  • 0.3 mg/ml glutamine
  • 0.1 mM BME,
  • 1 mM Nonessential amino acids
  • 10% serum
  • LIF
  • Sterile filter final mixture (0.2 mu;m filter)
• Gelatin
  • 0.1% TC-grade gelatin prepared in H₂O
  • Autoclave mixture and allow to cool before use