20.109(F09): TA notes for module 1

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DNA Engineering Module

Current: 20.109(F09)
20.109(F08) archive)
BE.109(S06) (archive)
20.109(S08) (archive)

General notes

Before the term:

  • Pre-run entire cloning procedure (Day 1-4) and save materials in case something goes wrong in the student's experiments
  • A plan for preparing the high volume of cell culture must be determined well in advance.
  • Reserve FACS time

Plasmids used: Stocks were prep'd by BioPioneer for F08 and F09 version of class.

  • pCX-EGFP: stock concentration = 0.55ug/ul
    • from strain NB124
    • contains full-length EGFP gene
    • Amp resistant
    • f1 ori
    • SV40 ori
    • For PCR (Day 1), dilute stock to about 100 ng/ul
    • For Transformation (Day 4), dilute stock to about 50 ng/ul
  • pCX-NNX: stock concentration = 0.5 ug/ul
    • from strain NB137
    • 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 stock concentration = 0.3 ug/ul
    • from strain NB168
  • pCX-EGFP with 5' deletion stock concentration = 0.55ug/ul
    • from strain NB153

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

Day of Lab:

  • No TA quiz needed today (lab practical will be taken instead).

Instructor's Bench: (not needed until near the end of lab)

  • Ice bucket
  • PCR Master Mix (thawed on ice only just before first group needs it at end of lab)
  • Sterile H20, 1 ml aliquot/group
  • Primers for PCR= D32N-fwd: NO234, D32N-rev: NO235, 100 pmol/ul
  • Template for PCR= pCX-EGFP 100 ng/ul
    • pCX-EGFP has been prepared from NB124. For PCR, dilute stock to about 100 ng/ul in sterile H2O.
  • Beaker with PCR tubes
  • PCR chill racks from -20° (1 per group)

PCR: Check the PCR machine has proper protocol under NK1:

  • Step 1: 94C 4min
  • Step 2: 94C 1 min
  • Step 3: 55C 1 min
  • Step 4: 72C 1 min
    • Repeat steps 2-4 34 more times
  • Step 5: 72C 10min
  • Step 6: 4C hold forever
    • Remember to freeze PCR products when they are ready.

Day 2

Materials required:

  1. Qiagen QIAquick PCR purification kit
    • from VWR # 28104, 50 rxns
    • 1 rxn per group
  2. pCX-NNX, 20 μL of 0.5 ug/ul stock per group
  3. NEB2 Buffer (~5 μL used per group)
  4. EcoRI and XbaI enzymes (~2 μL used per group)

Day of Lab:

  • Need to write/give/grade quiz
  • Thaw PCR products and leave at RT on instructor's bench
  • Thaw pCX-NNX and aliquot so there is at least 20 ul/group. Leave aliquot in ice bucket on instructor's bench.
  • 1 ice bucket with ice for each group at their benches

Looking ahead at TC
Check stocks for TC media

  • Filtered DMEM Complete
    • 500mL DMEM (high glucose)
    • 50mL serum (FBS from Atlanta Biological)
    • 5mL P/S/G
    • 1mL BME
    • 5mL NEAA
    • LIF
  • Filtered DMEM Pre-Txn Media
    • 500mL DMEM (highglucose)
    • 50mL serum (FBS)
    • 5mL 100XS glutamine
    • 1mL BME
    • 5mL NEAA

Day 3

Materials required:

  1. Qiagen QIAquick gel extraction kit
    • 2 extractions per group
    • kit from VWR # 28704, 50 rxns
  2. isopropanol (one 500 μL aliquot/group)
  3. sterile DI water (one 500 μL aliquot/group)
  4. loading dye for agarose gel electrophoresis (one 35 μL aliquot/group)
  5. 1% agarose gels prepared in 1X TAE 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
    • Groups will also run the original stock of pCX-NNX (make mix for all to use so each group loads 10 ul (= 2 μL of the stock, 8 μL water and 2 μL loading buffer for each group)
  6. Confirm digital camera OK
  7. Minigel (1%) to check recovery of student's fragments after class

Day of Lab:

  • Need to write/give/grade quiz
  • Run purified products (2 per group) on an agarose gel at the end of the day - post photograph.
  • Freeze DNA at end of day.

Looking ahead at TC

  • Cells should be growing
    • Will need one 60mm dish of MES per person on Day 6
    • Will need one 24 well dish of MES per group on Day 7

Day 4

Materials required:

  1. Buy fresh ligation buffer, T4 ligase from NEB
  2. 3 M sodium acetate (one 100 μL aliquot/group)
  3. Yeast tRNA (one 25 μL aliquot/group)
  4. Cold 100% ethanol (one 1 mL aliquot/group)
  5. Cold 70% ethanol (one 3 mL aliquots/group)
  6. Sterile DI water (one 100 μL aliquot/group)
  7. pCX-EGFP transformation control (dilute stock to 50 ng/ul and aliquot so at least 1 μL is available per group)
  8. competent cells
    • XL1-Blue from Stratagene, cat # ?200249?
    • 1 tube per group
  9. LB+Amp plates, 5 per group + spares
    • may get ~ 40 plates/Liter LB
  10. LB liquid medium, stored at RT
  11. Amp stock, stored at 4°
  12. Check spreaders, refill EtOH beakers and alcohol burners


Day of Lab:

  • Need to write/give/grade quiz

Students Benchtop

    • LB (~5 ml in 15mL conical tube/group)
    • LB+Amp plates (5 per group, so at least 30 per lab)
    • 1mL 3M NaAc (RT)
    • Ice buckets with ice
    • 25 ul tRNA in ice
    • 1 ml 100% EtOH in ice
    • 3 ml 70% EtOH in ice

Instructors Benchtop

    • Ice bucket--these materials should be thawed just before needed by students and kept on ice
      • Ligase and ligation buffer
      • Supercomp cells (need ~200 ul/group, so one tube of cells needed per group)
      • pCX-EGFP transformation control (50 ng/ul)
    • Spreaders, EtOH beakers and alcohol burners (these may need to be refilled with EtOH)

Post Lab Prep

  • The night before Day 5: pick colonies for overnight cultures (3 candidates per group, plus 1 pCX-NNX) into 2.5 ml LB+Amp. Grow 37° on roller wheel.


Day 5

Materials required:

  1. Miniprep solutions
  2. Restriction enzymes and buffers
  3. 37° incubator

Student Benchtop

  • Liquid cultures (3 candidates, 1 pCX-NNX control per group).
  • Miniprep solutions
    • Soln I (one 500 μL aliquot/group)
    • 2% SDS (one 600 μL aliquot/group)
    • 0.4M NaOH (one 600 μL aliquot/group)
    • Soln III (one 800 μL aliquot/group)
    • 100 % ethanol (one 5 mL aliquot/group)
    • 70 % ethanol (one 3 mL aliquot/group)
    • Sterile DI water (one 250 μL aliquot/group)

Instructor Benchtop

  • Ice bucket
  • Midway through class will need EcoRV, XbaI, BamHI, XhoI and perhaps other enzymes
  • 10X NEB buffers 1, 2, 3, 4 thawed

Post Lab Prep

  • Ensure digests are frozen at end of day

Looking ahead at TC
Next time: TC will need one 60mm dish of confluent J1s PER STUDENT (not per group!) plus two extras for demo.

Day 6

This lab can be chaotic since essentially two labs are running at once: half the students will be in the TC facility splitting cells and the other half will be in the main lab running a gel. Halfway through they will switch.

Materials required:

  1. Main lab:
    • Agarose gels, 1XTAE, 15 well combs, 2 combs/gel. Two groups can share one gel.
  2. TC reagents
    • One 60 mm dish of MES cells near confluence per student
    • 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)
    • 12 six-well dishes
  3. Canisters autoclaved Pasteur pipets
  4. Charged Pipet-Aids

Looking ahead at TC

24 hours before Day 7 need to plate 24 well dishes (1 per pair) in DMEM Pre-Txn Media

From confluent 100mm dish, wash with PBS, add 2 mL trypsin in hood, aspirate incubate(dry) for 10', triterate with 5mL Pre-txn media. Add 0.7mL of this suspension to 14mL pre-txn media (~1:20), place 0.5 ml/well of pre-gelatinized 24 well dish overnight

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)
  4. Sterile eppendorf tubes

Instructors Bench

  • Plasmids for Lipfoection
  • pCX-EGFP
  • pCX-del5
  • pCX-del3
  • Also need
  • pCX-del3 cut with PmeI (blunt)
  • pCX-del3 cut with BamHI (4 bp)
  • pCX-del3 cut with N.BbvcIB (14bp)

In pre-run about 2ug of pCX-del3 was cut for each and recovered 30ul of 0.05ug/ul cut DNA after gel purification

  • (5ul in 500 to measure conc. IOD260 = 50ug/ml

It will be easiest to dilute all DNAs to approx same concentration (pCX-EGFP and pCX-del5 to 0.25ug/ul and all pCX-del3s to 0.05ug/ul would be ideal). Be sure to use STERILE WATER and TC pipets... in Spring of 04 there was terrible contamination of these transfections.

Prep Work For today's lab need 24 well dish (1 per pair) in media WITHOUT antibiotics

Tomorrow will need to remove media and add 1mL fresh Complete DMEM

Confirm FACS time for Day 8 (3 horus starting at 1:30pm)

Day of Lab:

  • Quiz (prepared by TA).

Day 8

FACS time: 1:30-4:30pm

The day will be a repetitive one at the FACS facility. Each group with come with 16 samples to collect. Print out two copies of data for each group, one for them and one for the lab's notes. MES cell culture is done after today!! w00t

Materials required:

  1. TC reagents
    • PBS (20 mL per group)
    • Trypsin (4 mL per group)
    • OptiMEM (2 mL per group)
  2. FACS tubes
    • 16 per group, plus extras
    • BD Falcon tubes only, VWR cat # 60819-310 (1000x) or 60819-295 (500x)

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°.
  2. For LB-Amp plates, add Amp (2 ml / L) after autoclaving, once the mixture has cooled down to warm but not hot.
  3. Amp: 100 mg/ml in H20. 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 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
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